Soluble tcr molecules and methods of use

ABSTRACT

Disclosed are compositions and methods for detecting cells or tissue comprising a peptide antigen presented in the context of an MHC or HLA complex. The invention has a wide range of applications including providing a highly sensitive method for detecting cancer cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/182,888, filed Feb. 18, 2014, which is a continuation of U.S.application Ser. No. 10/985,271, filed Nov. 10, 2004, which claims thebenefit of U.S. Provisional Application No. 60/518,790, filed Nov. 10,2003, each of which is incorporated herein by reference in its entirety.

STATEMENT OF U.S. GOVERNMENT INTEREST

Funding for the present invention was provided in part by the Governmentof the United States by virtue of Grant Nos.: 1R43CA88615-01 and1R43CA105816-01 from the National Institutes of Health. Accordingly, theGovernment of the United States has certain rights in and to theinvention claimed herein.

FIELD OF THE INVENTION

The invention features compositions and methods for detecting cells ortissue comprising a peptide antigen presented in the context of an MHCor HLA complex. The invention has a wide range of applications includingproviding a highly sensitive method for detecting cancer cells.

BACKGROUND

There is increasing recognition that immunotherapy is a promisingapproach to treat cancer. Various strategies have been proposedincluding treatment with cytokines such as interleukin-2 (IL-2). IL-2impacts various immune cell types including T and B cells, monocytes,macrophages, lymphokine activated killer cells (LAK) and NK cells [10,40].

There have been proposals to concentrate cytokines at the site of tumorsto help increase efficacy. Typical methods include direct injection ofthe cytokine or gene encoding same into the tumor, or targeted deliveryof the cytokine by fusing it to a tumor antigen specific antibody [20].However, these methods have drawbacks.

For example, most direct injection methods are difficult to useespecially at early stages of cancer when tumors are typically small(micrometastases). Moreover, such methods are usually labor-intensivewith little guarantee of therapeutic success. This makes treatment oflarge patient populations impractical and costly.

Antibody-cytokine fusion constructs have been used in an approach totreat cancer. However, the methods are limited to the extent that theantibody has a limited binding spectrum. That is, the antibodies canonly recognize certain cell surface antigens. Unfortunately, many tumorantigens are not displayed appropriately for antibody recognition,thereby limiting potential of antibody based approaches. Moreover, thereare reports that many tumor specific antigens are derived from aberrantexpression of cell type specific proteins. These may exist only with asmall number of tumor types. This drawback limits the potential ofantibody based therapies even further.

The p53 protein is an intracellular tumor suppressor that has beenreported to act by arresting abnormal cells at the G1/S phase of thecell cycle. Over expression of the protein is believed to be asignificant tumor marker for a large number of human malignancies andthere is recognition that it is a good target for broad spectrumtargeted tumor immunotherapy. The p53 protein is usually displayed onthe cell surface in the context of major histocompatibility complexproteins (MHC). Such protein complexes are known to be the bindingtargets of T-cell receptors (TCRs). [49].

There have been attempts to use certain TCRs to detect MHC/peptidecomplexes containing peptide (Epel et al., 2002; Holler et al., 2003;Lebowitz et al., 1999; Plaksin et al., 1997; Wataya et al., 2001;O'Herron et al., 1997). However, these and related methods havesignificant shortcomings.

For instance, many of the methods require that TCR constructs bemultimerized (i.e., designed to have multiple TCR copies) presumably toenhance peptide antigen binding with peptide antigen artificially.Target (antigen presenting) cells are often manipulated by the methodsto express relatively large amounts of peptide antigen. Sometimes thedensity of peptide antigen is as high as 10⁴ to 10⁵ complexes per cell(Wataya et al., 2001). Such a high peptide antigen density is believedto facilitate binding and detection by the TCRs. However, these levelsof peptide antigen are artificial and typically much greater than thelevel of MHC/peptide complexes that include most tumor-associatedantigens (TAAs). For some TAAs, less than about 50 HLA/peptide complexesper cell are present (Pascolo et al., 2001; Schirle et al., 2000). Thus,there has been recognition that the prior methods are not sensitiveenough to detect most if not all TAAs.

There have been attempts to use certain TCRs to detect cells expressingparticular peptide antigens. Like many antibody based methods, theseapproaches have either lacked enough sensitivity to detect peptideantigen or failed to detect such antigen completely.

For example, Holler et al. (2003) reported the development of certainsoluble TCRs that were reported to react with MHC/peptide complexes.Although the TCRs were able to detect antigen with cells artificially“loaded” with the antigen, the molecules were unable to detectendogenous antigen on tumor cells. Holler et al. concluded that when theantigen is present at a density of less than 600 copies per cell, TCRbased methods are not sensitive or reliable enough to detect antigen.

Particular TCR based methods have been used to detect viral peptides inthe context of MHC molecules. (Strominger, et al., WO9618105). However,these and related methods suffer from drawbacks. For instance, there isgeneral recognition that viral infection often produces exceptionallyhigh densities of MHC/peptide complexes, typically approachingfrom >1000 to >10⁵ complexes per cell. See Herberts et al., 2001; vanEls et al., 2000. Thus like most other peptide antigen detectionmethods, TCR based approaches to detect viral antigens have so farrelied on the relatively large number of antigen targets to drive thedetection method.

Although some TCR based methods have been used to detect relativelylarge amounts of peptide antigen, it is less certain if the methods willwork when the TCR is fused to other molecules such as a cytokine, animmunoglobin domain such as IgG1, biotin or streptavidin. That is, it isnot certain how the resulting fusion molecule will impact the TCRpeptide binding groove particularly when low densities of TAA need to beanalyzed. Small distortions in the TCR peptide binding groove, while notnecessarily problematic when relatively large amounts of peptide antigenare to be analyzed, could reduce TAA binding specificity andselectivity. Even small changes in the TCR peptide binding groovefunction could jeopardize detection of cancer cells that express low TAAdensities.

It would be useful to have methods for detecting TAAs that aresensitive, selective and reproducible especially when the peptideantigens are present in low densities. It would be especially useful ifsuch methods could be used with a variety of soluble TCRs includingmolecules such as those fused to a detectable label or a cytokine.

SUMMARY OF THE INVENTION

The invention generally features a method for detecting cells or tissuecomprising a peptide antigen presented on the cells or tissue in thecontext of an MHC or HLA complex. In one embodiment, the inventionincludes at least one and preferably all of the following steps:

-   -   a) contacting the cells or tissue with at least one soluble TCR        molecule or functional fragment thereof under conditions that        form a specific binding complex between the presented peptide        antigen and the soluble TCR or fragment,    -   b) washing the cells or tissue under conditions appropriate to        remove any soluble TCR molecule or fragment not bound to the        presented peptide antigen; and    -   c) detecting the specific binding complex as being indicative of        cells or tissue comprising the presented peptide antigen.

In preferred practice, the invention is used to detect an amount ofpeptide antigen on the cells or tissue that is less than about 100,000copies, preferably less than about 1000 copies such as about 100 toabout 800 copies.

Use of the invention has several advantages. For instance, the inventionis highly sensitive and can be used to detect and optionally quantitatevery low-density MHC/peptide complexes including those containingendogenous peptide, more particularly tumor-associated peptide antigenspresented on unmanipulated tumor cells. In contrast, prior methods fordetecting MHC/peptide complexes are reported to be capable of detectingrelatively higher density complexes.

Additionally, the invention can be used to detect and optionallyquantitate fixed cells and tissues such as those routinely found inhistoarrays, for example tumor histoarrays. The ability to detectMHC/peptide complexes (sometimes called “staining”) is advantageous,especially in clinical or other medical settings where it is typicalpractice to fix cells, tissues or other biological samples taken frompatients. In contrast, many prior TCR-based detection methods are notable to accommodate fixed tissue since noncovalently associated peptideis routinely lost during the tissue processing steps.

The invention provides still further advantages. For instance, themethods are intended to be flexible and compatible with use of monomericand/or multimeric soluble TCR molecules. Unfortunately, past practicehas relied heavily on use of multimeric TCRs which has limitedflexibility and sensitivity. In particular, such multimeric TCRs may bedifficult to use for in vivo imaging due to their potential forbreakdown or aggregation, lack of accessibility to the target site,increased immunogenicity and clearance.

Practice of the invention addresses a long-felt need in the field byproviding an ability to detect endogenous peptide antigen presented inthe context of the MHC/peptide complex on the surface of cells. Themethod has a variety of important uses such as helping to monitor cellactivity, pathology and infection. For example, detection of endogenoustumor-associated peptide antigens on cells or tissues by the inventioncan provide a means of detecting and optionally quantitating thepresence/extent of a cancer. Past practice has often relied onantibodies as a diagnostic tool to detect protein antigens on thesurface of cancer cells. However, antibodies typically are limited indetection of cell-membrane proteins. In addition, detection withantibodies is often compromised by antigen shedding or secretion of theantigenic protein into the circulation. Antibodies also have limitedtarget recognition. Practice of the invention avoids these and otherdifficulties by providing a sensitive and reliable detection method thatuses soluble TCRs and fragments thereof to detect target peptideantigens.

Such uses and advantages of the invention can be employed to detectpeptide antigen in a variety of settings including in vivo (e.g., as animaging or diagnostic method) or in vitro (e.g., in a histoarray or FACSanalysis).

Other aspects of the invention are discussed infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B are drawings showing the schematic structure (FIG. 1A) andthe amino acid sequence (FIG. 1B) of a 264scTCR/IL-2 fusion protein (SEQID NO: 16). (G₄S)₄ linker disclosed as SEQ ID NO: 17.

FIG. 2 is a representation of a sizing gel showing production of264scTCR/IL-2 fusion protein in transfected CHO cells.

FIG. 3A, FIG. 3B, and FIG. 3C are graphs showing MHC/peptide bindingability of the TCR portion of 264scTCR/IL-2 fusion protein.

FIG. 4A-FIG. 4B are graphs showing IL-2 receptor binding ability of theIL-2 portion of 264scTCR/IL-2 fusion protein.

FIG. 5A-FIG. 5B are graphs showing biological activity of 264scTCR/IL-2fusion protein.

FIG. 6A, FIG. 6B, FIG. 6C, and FIG. 6D are graphs showing conjugation ofCTLL-2 cells with peptide-loaded T2 cells mediated by 264scTCR/IL-2fusion protein.

FIG. 7A, FIG. 7B, and FIG. 7C are graphs showing serum half life of264scTCR/IL-2 fusion protein.

FIG. 8 is a graph showing tumor cell staining with 264scTCR/IL-2 fusionprotein.

FIG. 9 is a graph showing anti-tumor effect of 264scTCR/IL-2 fusionprotein.

FIG. 10A and FIG. 10B are graphs showing staining of T2 cells loadedwith non-specific p53 peptide (FIG. 10A) or specific p53 peptide (FIG.10B) with 264scTCR reagents.

FIG. 11A, FIG. 11B, FIG. 11C, and FIG. 11D are graphs showing stainingof tumor cells with various 264scTCR reagents and secondary reagents.

FIG. 12A and FIG. 12B are graphs showing staining of fixed A375 (FIG.12A) or T2 cells (FIG. 12B) with 264scTCR/IgG1 and CMVscTCR/IgG1(control) reagents, with and without the addition of competing solublepeptide-MHC molecules (labeled 264-Tet).

FIG. 13 is a series of photomicrographs showing staining patterns offixed tumor cell types (A375, HT29 and Saos2) for A2 and p53 antigensand with 264scTCR/IgG1 and CMVscTCR/IgG1 fusion proteins.

FIG. 14 is a graph showing quantitative staining of T2 cells reactedwith 264scTCR/BirA tetramers.

FIG. 15 is a graph showing quantitative staining of T2 cells reactedwith 264scTCR/IgG1 fusion protein.

FIG. 16 is a graph showing numbers of complexes per cell with increasingamounts of loaded p53 peptide, for cells reacted with 264scTCR/BirAtetramer or 264scTCR/IgG fusions.

FIG. 17A and FIG. 17B are graphs showing quantitative staining of A375tumor cells reacted with 264scTCR/BirA tetramers (FIG. 17A) or264scTCR/IgG1 fusion protein (FIG. 17B).

FIG. 18 is a graph showing quantitative staining (number of complexesper cell) of three tumor cell lines reacted with 264scTCR/BirAtetramers.

FIG. 19 is a graph as in FIG. 18 showing quantitative staining of threetumor cell lines reacted with 264scTCR/IgG1 fusion protein.

FIG. 20 is three photomicrographs showing fixed sections of A375 tumorstained with secondary antibody, CVMscTCR/IgG1 (control) or 264TCR/IgG1fusion protein, at 200×.

FIG. 21 is three photomicrographs showing tumor sections as in FIG. 20,at higher magnification (400×).

DETAILED DESCRIPTION

As discussed, the invention generally involves a method for detectingcells or tissue comprising a peptide antigen presented on the cells ortissue in the context of an MHC complex. In one embodiment, theinvention includes contacting the cells or tissue with at least onesoluble TCR molecule or functional fragment thereof under conditionsthat form a specific binding complex between the presented peptideantigen and the soluble TCR or fragment; washing the cells or tissueunder conditions appropriate to remove any soluble TCR molecule orfragment not bound to the presented peptide antigen; and detecting thespecific binding complex as being indicative of cells or tissuecomprising the presented peptide antigen.

In general, preparation of the present soluble TCRs can be accomplishedby procedures disclosed herein and by recognized recombinant DNAtechniques. For example, preparation of plasmid DNA, DNA cleavage withrestriction enzymes, ligation of DNA, introduction of DNA into a cell,culturing the cell, and isolation and purification of the expressedprotein are known techniques. See generally Sambrook et al. in MolecularCloning: A Laboratory Manual (2d ed. 1989); and Ausubel et al. (1989),Current Protocols in Molecular Biology, John Wiley & Sons, New York.

The general structure of a variety of soluble TCR constructs and methodsof making and using same have been disclosed in pending U.S. applicationSer. Nos. 08/813,781 and 08/943,086.

For instance, a particular soluble TCR is a heterodimer in whichtransmembrane sequence in at least one of and preferably both of the Vchains has been deleted. However for convenience, it will often bepreferred to use single-chain (“sc-”) constructs such as those reportedby the pending Ser. Nos. 08/813,781 and 08/943,086 applications.

Briefly stated, a single-chain (“sc-”) TCR molecule includes V-α and V-βchains covalently linked through a suitable peptide linker sequence. Forexample, the V-α chain can be covalently linked to the V-β chain througha suitable peptide linker sequence fused to the C-terminus of the V-αchain and the N-terminus of the V-β chain. The V-α and V-β chains of thesc-TCR fusion protein are generally about 200 to 400 amino acids inlength, preferably about 300 to 350 amino acids in length, and will beat least 90% identical, and preferably 100% identical to the V-α and V-βchains of a naturally-occurring TCR. By the term “identical” is meantthat the amino acids of the V-α or V-β chain are 100% homologous to thecorresponding naturally-occurring TCR V-β or V-α chains.

As disclosed in the Ser. No. 08/943,086 application, the V-α chain ofthe sc-TCR molecule can further include a C-β chain or fragment thereoffused to the C-terminus of the V-β chain. Further, the V-α chain caninclude a C-α chain or fragment thereof fused to the C-terminus of theV-α chain and the N-terminus of the peptide linker sequence. Generally,in those fusion proteins including a C-β chain fragment, the fragmentwill have a length of approximately 50 to 130 amino acids and willusually not include the last cysteine residue (at position 127 in themouse or at position 131 in the human) of the C-β chain. For thosefusion proteins comprising a C-α chain, the length can vary betweenapproximately 1 to 90 amino acids (i.e. the C-α chain up to but notincluding the final cysteine). For example, in one embodiment, thefusion protein includes a C-α chain fragment between about 1 to 72 aminoacids starting from amino acid 1 to 72. In another embodiment, the C-αchain fragment is between about 1 to 22 amino acids starting from thefirst amino acid to 22 (leucine). The C-α chain fragment typically doesnot include any cysteine resides except the C_(∝90) variant whichincludes two cys residues and the C_(∝72) variant which includes one cysresidue. In most cases, choice of Cα and Cβ chain length will be guidedby several parameters including the particular V chains selected andintended use of the soluble fusion molecule.

As further disclosed by the Ser. No. 08/943,086 application, additionalsc-TCR proteins of the invention include e.g., two peptide linkersequences, where the first peptide linker sequence is fused between theC-terminus of the V-α chain and the N-terminus of the V-β chain. TheC-terminus of the V-β chain can be fused to the N-terminus of a C-βchain fragment. The second peptide linker is then fused to theC-terminus of the V-β chain or C-β chain fragment or, if desired, to atag molecule as explained below. In other illustrative embodiments,sc-TCR proteins can be made by fusing the V-β chain to the V-α chainthrough a suitable peptide linker in which the C-terminus of the V-βchain or C-β chain fragment thereof and the N-terminus of the V-α chainare covalently linked.

A soluble TCR protein according to the invention can include one or morefused protein tags. In embodiments in which such tags are “detectable”,the soluble TCR will be referred to as being “detectably labeled”. Forexample, with respect to a soluble fusion protein, a protein tag can befused to the C-terminus of the sc-TCR V-β chain (or C-β chain fragment).If desired, such soluble TCR proteins can be fused to immunoglobinchains as has been reported by the pending Ser. No. 08/943,086application, and further illustrated in Examples below.

Preferred soluble fusion proteins for use with the invention are fullyfunctional and soluble. By the term “fully functional” or similar termis meant that the fusion protein specifically binds ligand. Assays fordetecting such specific binding are disclosed herein and includestandard immunoblot techniques such as Western blotting. Functionalfragments of such soluble TCRs are able to bind antigen with at least70% of the affinity of the corresponding full-length TCR, preferablyabout 80% to 90% or more as determined by Western blot or Surface PlasmaResonance analysis.

The nucleic acid and protein sequences of suitable TCR chains have beendisclosed. See e.g., Fundamental Immunology, (1993) 3^(rd) Edi. W. Paul.Ed. Rsen Press Ltd. New York; and Kabat, E. A., et al., (1991) Sequencesof Proteins of Immunological Interest (5^(th) Ed.) Public HealthServices, National Institutes of Health. See also the pending Ser. Nos.08/813,781 and 08/943,086 applications as well as the Examples thatfollow.

In a particular embodiment of the invention, the method further includescontacting the cells or tissue with at least one blocking agent. Thecontacting step can be performed at any point in the method includingbefore, during or after step a) to reduce non-specific binding betweenthe soluble TCR or fragment and the cells. The invention is compatiblewith use of nearly any standard blocking agent such as peroxide, serumprotein, antibody or an antigen-binding fragment thereof.

In certain embodiments, it will often be useful to confirm the bindingspecificity of the TCR to the MHC complex on the cells or tissues to bedetected. In such instances, the invention can further includecontacting the specific complex (formed between the soluble TCR and theMHC complex residing on the cells or tissue) with a competing MHC (orHLA) molecule or fragment thereof under conditions that compete with andspecifically bind the soluble TCR or fragment bound to the complex. Avariety of suitable MHC molecules have been disclosed.

In one embodiment of the method, specific binding of the soluble TCR orfragment is reduced or essentially eliminated by the addition of acompeting MHC molecule or fragment thereof, such that the soluble TCR orfragment is bound to the competing MHC molecule or fragment thereof toform a competition complex. In one particular embodiment of the method,the competing MHC molecule is added at a range of concentrations betweena 0.01 to 1000 fold, or preferably a 1 to 100 fold, molar excess overthe soluble TCR. In another embodiment, the competing MHC molecule isadded at a single concentration (i.e. 1-fold, 10 fold, or 100-fold molarexcess over the soluble TCR) sufficient to reduce specific binding ofthe soluble TCR. If desired, that competition complex can be detectedand binding specificity of the MHC molecule or the soluble TCRdetermined by one or a combination of conventional strategies.Particular MHC molecules or fragments can be single-chain but in mostinstances will be soluble heterodimeric molecules such as thosedisclosed in U.S. Pat. Nos. 5,869,270; 6,309,645; and pendingapplication Ser. No. 09/848,164. See also PCT application PCT/US95/09816for additional disclosure, as well as the Examples provided below.Typical MHC molecules or fragments will be loaded with peptide antigen.

See also the following published U.S. patent applications for disclosurerelating to other soluble TCR and MHC molecules that can be used topractice the invention: 20020198144; 20020091079; 20020034513;20030171552; 20030144474; 20030082719; and references cited therein.

In a typical method in which confirmation of binding specificity isdesired, the TCR molecule or fragment is detectably-labeled with one ormore tags. Suitable tags include EE or myc epitopes which arespecifically bound by commercially available monoclonal antibodies. Ingeneral, a wide variety of epitopes capable of being specifically boundby an antibody, e.g., a monoclonal antibody, are capable of serving as aprotein tag. Other suitable synthetic matrices include those with abound antibody capable of specifically binding the molecules. Furthertags include those with an enterokinase, Factor Xa, snake venom orthrombin cleavage site. See e.g., published PCT application WO 96/13593.

Other suitable tags for detectably-labeling the TCR molecules orfragments include biotin, streptavidin, a cell toxin of, e.g., plant orbacterial origin such as, e.g., diphtheria toxin (DT), shiga toxin,abrin, cholera toxin, ricin, saporin, pseudomonas exotoxin (PE),pokeweed antiviral protein, or gelonin Biologically active fragments ofsuch toxins are well known in the art and include, e.g., DT A chain andricin A chain. Additionally, the toxin can be an agent active at thecell surface such as, e.g., phospholipase enzymes (e.g., phospholipaseC). See e.g., Moskaug, et al. J. Biol. Chem. 264, 15709 (1989); Pastan,I. et al. Cell 47, 641, 1986; Pastan et al., Recombinant Toxins as NovelTherapeutic Agents, Ann. Rev. Biochem. 61, 331, (1992); “ChimericToxins” Olsnes and Phil, Pharmac. Ther., 25, 355 (1982); published PCTapplication no. WO 94/29350; published PCT application no. WO 94/04689;and U.S. Pat. No. 5,620,939 for disclosure relating to making and usingproteins comprising effectors or tags. An example of a tag that performsa biotin acceptor function is a BirA tag, as described in Beckett, D. etal. Protein Sci. 1999 April; 8(4):921-9. As further described inExamples below, a BirA tag sequence can be included in a TCR molecule topromote biotinylation of the protein. Further, a tag can be achemotherapeutic drug such as, e.g., vindesine, vincristine, vinblastin,methotrexate, adriamycin, bleomycin, or cisplatin.

Additionally, a tag can be a radionuclide or chelate, suitable fordiagnostic or imaging studies such as iodine-131, yttrium-90,rhenium-188, iodine-123, indium-111, technetium-99m, gallium-67,thallium-201, or bismuth-212. Among the radionuclides used,gamma-emitters, positron-emitters, x-ray emitters andfluorescence-emitters are suitable for localization, while beta-emittersand alpha-emitters may also be used. Other suitable radioisotopes forthe methods of the present invention include but are not limited to,cadmiun-109, actinium-225, actinium-227, astatine-211, iodine-125,iodine-126, iodine-133, dysprosium-165, dysprosium-166, bismuth-212,bismuth-213, bromine-77, indium-113m, gallium-67, gallium-68,ruthenium-95, ruthenium-97, ruthenium-101, ruthenium-103, ruthenium-105,mercury-107, mercury-203, rhenium-186, rhenium-188, tellurium-99m,tellurium-121m, tellurium-122m, tellurium-125m, thulium-165,thulium-167, thulium-168, fluorine-18, silver-111, platinum-197,palladium-109, copper-67, phosphorus-32, phosphorus-33, yttrium-90,scandium-47, samarium-153, lutetium-177, rhodium-105, praseodymium-142,praseodymium-143, promethium-149, terbium-161, holmium-166, gold-198,gold-199, cobalt-57, cobalt-58, chromium-51, iron-59, selenium-75, andytterbium-169. Preferably the radioisotope will emit in the 10-5,000 keyrange, more preferably 50-1,500 key, most preferably 50-500 key.

Suitable positron emitters and other useful radionuclides include, butare not limited to, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁵¹Mn, ⁵²Fe, ⁵⁵CO, ⁶⁰CU, ⁶¹CU,⁶²CU, ⁶⁴CU, ⁶²Zn, ⁶³Zn, ⁷⁰As, ⁷¹As, ⁷²As, ⁷⁶Br, ⁸²Rb, ⁸⁶Y, ⁸⁹Zr, ⁹⁴mTc,¹¹⁰In, ¹²⁰I, ¹²⁴I, ¹²²Xe, ¹²⁸Ba, ¹³¹Ba, ⁷Be, ²⁰⁴Bi, ²⁰⁵Bi, ²⁰⁶Bi, ¹⁴C,³⁶Cl, ⁴⁸Cr, ⁵¹Cr, ¹⁵⁵Eu, ¹⁵³Gd, ⁶⁶Ga, ⁷²Ga, ³H, ^(115m)In, ¹⁸⁹Ir,^(191m)Ir, ¹⁹⁴Ir, ⁵⁵Fe, ⁵⁹Fe, ^(119m)Os, ⁴²K, ²²⁶Ra, ¹⁸⁶Re, ¹⁸⁸Re,^(82m)Rb, ⁴⁶Sc, ⁴⁷Sc, ⁷²Se, ¹⁰⁵Ag, ²²Na, ²⁴Na, ⁸⁹Sr, ³⁵S, ³⁸S, ¹⁷⁷Ta,⁹⁶Tc, ²⁰¹Tl, ²⁰²Tl, ¹¹³Sn, ¹¹⁷msn, ¹²¹Sn, ¹⁶⁶Yb, ¹⁷⁴Yb, ⁸⁸Y, ⁹⁰Y, ⁶²Znand ⁶⁵Zn.

Suitable chelates include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA),1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA),1-substituted 1,4,7,-tricarboxymethyl 1,4,7,10 teraazacyclododecanetriacetic acid (DO3A), ethylenediaminetetraacetic acid (EDTA), and1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA).Additional chelating ligands are ethylenebis-(2-hydroxy-phen-ylglycine)(EHPG), and derivatives thereof, including 5-C1-EHPG, 5Br-EHPG,5-Me-EHPG, 5t-Bu-EHPG, and 5 sec-Bu-EHPG; benzodiethylenetriaminepentaacetic acid (benzo-DTPA) and derivatives thereof, includingdibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzylDTPA; bis-2 (hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) andderivatives thereof; the class of macrocyclic compounds which contain atleast 3 carbon atoms, more preferably at least 6, and at least twoheteroatoms (O and/or N), which macrocyclic compounds can consist of onering, or two or three rings joined together at the hetero ring elements,e.g., benzo-DOTA, dibenzo-DOTA, and benzo-NOTA, where NOTA is1,4,7-triazacyclononane N,N′,N″-triacetic acid, benzo-TETA, benzo-DOTMA,where DOTMA is 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyltetraacetic acid), and benzo-TETMA, where TETMA is1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic acid);derivatives of 1,3-propylenediaminetetraacetic acid (PDTA) andtriethylenetetraaminehexaacetic acid (TTHA); derivatives of1,5,10-N,N′,N″-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM) and1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl) aminomethylbenzene (MECAM).

Other suitable tags include polyhistidine, fluorescent label,chemiluminescent label, nuclear magnetic resonance active label,chromophore label, positron emitting isotope detectable by a positronemission tomography (“PET”) scanner, enzymatic markers such asbeta-galactosidase and peroxidase including horse radish peroxidase, ananoparticle, a paramagnetic metal ion, a contrast agent or an antigenictag.

A suitable fluorescent label could include, but is not limited to, a¹⁵²Eu label, a fluorescein label, an isothiocyanate label, a rhodaminelabel, a phycoerythrin label, a phycocyanin label, an allophycocyaninlabel, an o-phthaldehyde label, a Texas Red label, a fluorescaminelabel, a lanthanide phosphor label, a fluorescent protein label, forexample a green fluorescent protein (GFP) label, or a quantum dot label.Examples of chemiluminescent labels include a luminal label, anisoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridinium salt label, an oxalate ester label, a luciferinlabel, a luciferase label, an aequorin label, etc.

Suitable paramagnetic metal ions include, but are not limited to, Mn²⁺,Cu²⁺, Fe²⁺, Co²⁺, Ni²⁺, Gd³⁺, Eu³⁺, Dy³⁺, Pr³⁺, Cr³⁺, Co³⁺, Fe³⁺, Ti³⁺,Tb³⁺, Nd³⁺, Sm³⁺, Ho³⁺, Er³⁺, Pa⁴⁺, and Eu²⁺.

Enzyme markers that may be used include any readily detectable enzymeactivity or enzyme substrate. Such enzymes include malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, alcoholdehydrogenase, aglycerol phosphate dehydrogenase, triose phosphateisomerase, peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholine esterase,luciferase, and DNA polymerase.

Suitable nanoparticles include, but are not limited to, solid colloidalparticles, dendrimers, liposomes, micelles, ceramic particles, aluminacapsules, emulsifying wax or Brij 72 particles, ferromagnetic particles,gold or silver particles, biodegradable particles comprisingpoly(lactic-co-glycolic) acid, polyglycolic acid, poly D- or L-lacticacid, polycaprolactone or serum albumin and particles comprisingpoly(vinyl pyrrolidone), polystyrene, polyacrylamide, or poly(butylcyanoacrylate) or derivative thereof. In some applications of theinvention, nanoparticles coated with agents such a polyethylene glycol,polysaccharide, polypeptides, lipids, silica, etc. can be used. Suchcoated nanoparticles may have improved absorbance, bioavailability,tissue distribution, tissue cross-reactivity, toxicity,pharmacokinetics/dynamics, or tumor localization. Methods for attachingtargeting ligands to nanoparticles have been described that can beapplied to soluble TCR-based reagents (see, for example, Nob et al.2004. J Pharm Sci. 93:1980-92).

The soluble TCRs of the invention include monomeric and multimeric TCRs.Multimeric TCR molecules include those in which the TCR protein is fusedto polypeptide domains or tags that facilitate multimerization. Suchdomains include immunoglobin, leucine zipper, helix-turn-helix, andbarrel-barrel motifs that facilitate protein dimerization. Such tagsinclude antibody-binding epitopes, streptavidin-binding peptides, 6×Hismotif, biotin ligase target motif, and the like. Multimeric TCRmolecules also include those generated through chemically crosslinkingreactive amino acids or polysaccharides. Such amino acids (orpolysaccharides) can be inherent in the TCR structure or can be addedthrough genetic modification. Multimeric TCRs also include thosegenerated through attachment to another molecule (or molecules) that mayor may not include a detectable label as described herein. Suchattachment molecules include streptavidin, biotin, antibodies, protein Aor scaffolds that include protein-, lipid- and polysaccharide-coated oruncoated beads, nanoparticles, solid-phase surfaces, arrays, matrices,as described. For example, in various embodiments in which thedetectable label is biotin, the method further comprises combining theTCR molecule with streptavidin to multimerize the TCR molecule.

It will be appreciated that any one of the tags disclosed herein can beused to detectablylabel the soluble TCRs used in the invention method,particularly to detect the cells or tissue expressing the peptideantigen of interest.

It is an object of the invention to provide peptide antigen detectionmethods that perform using cells or tissue contacted with a denaturingagent sufficient to “fix” the cells or tissue. Examples of such agentsare known in the field and include formaldehyde (formalin),glutaraldehyde, alcohols such as methanol, proponal, etc, and organicsolvents such as benzene and xylene. As has been discussed, it has beenfound that the invention methods do not substantially disturbinteraction between the MHC molecule on the cells and its cognatepeptide antigen even when the cells or tissue are fixed. Thus, theinvention can be used on fixed cells or tissue, thereby helping topreserve structural integrity and enhancing reliability of the method.

Accordingly, and in one embodiment, the invention further comprisescontacting the cells or tissue with at least one denaturing agent. Suchcontact can be performed at nearly any time including before step a) anddenaturing (fixing) the cells or tissue.

As also discussed, the invention is compatible with use of cells ortissue in an array such as what is referred to in the field as ahistoarray. That is, the invention has the sensitivity and reliabilityneeded to screen cell or tissue samples (such as those encountered in aclinic) in a repetitive fashion. Many such arrays are known in the fieldsuch as those described by U.S. Pat. Nos. 6,466,690; 4,384,193;6,602,661; 6,594,432; 6,566,063; 6,406,840; 6,246,785; and referencescited therein.

Accordingly, and in one embodiment, the method of the present inventionfurther includes placing a plurality of cells or tissue in an array.Preferably, such cells or tissues are known or suspected of including(or consisting of) tumor cells. The method can be performed in eachelement of the array comprising cells or tissue. If desired, the methodis performed substantially simultaneously in each element of the array.In one embodiment, the step c) of the method further includes scanningthe array and generating image signals indicative of presence of thespecific binding complex. If needed, that step can further includeoutputting the signals in real-time to a user and optionally indexingstored images of the image signal.

The invention can be used to detect a wide variety of peptide antigensincluding those referred to as tumor-associated peptide antigens orTAAs. Cells or tissues may be suspended, semi-suspended, or fixedaccording to the method.

As discussed, the soluble TCR molecule or fragment can include at leastone single-chain TCR or it may be a heterodimeric construct such asthose that have been manipulated recombinantly to remove transmembranedomains. See the pending Ser. Nos. 08/813,781 and 08/943,086applications as well as the Examples that follow. Such soluble TCRmolecules or fragments can be detectably labeled by one or a combinationof strategies as outlined herein including labeling with biotin,streptavidin, an enzyme or catalytically active fragment thereof,radionuclide, or a fluorescent, phosphorescent, or chemiluminescentmolecule. Examples include the well-known green (or red) fluorescentprotein or fragments thereof.

In certain embodiments, the soluble TCR is a single-chain TCR whichmolecule is covalently bound to at least one cytokine. Examples of suchcytokines include, but are not limited to, IL-2, colony stimulatingfactors such as GM-CSF, IFNγ, IFN-α and the like. As an example, thesoluble TCR molecule or fragment is a single-chain TCR that includes atleast one and preferably one covalently bound cytokine or fragmentthereof.

In certain other variations, the soluble TCR is a single chain TCR orfragment that includes at least one covalently bound immunoglobulindomain or fragment thereof. In some embodiments the single chain TCR orfragment is fused to sequence comprising an IgG1 domain or fragment.

In yet another embodiment, the MHC complex is HLA-A2 restricted.

It will often be useful to include a control with the method, forexample, by detecting any binding between the soluble TCR or fragment tocells that do not comprise the peptide antigen.

A particular peptide antigen for use with the invention includes p53 (aa149-157) or p53 (aa 264-272).

The present invention methods can be performed in vivo, ex vivo, or invitro.

For instance, HLA typing (see, e.g., A. K. Abbas, Cellular and MolecularImmunology, page 328 (W.B. Saunders Co. 1991) can be practiced with theinvention. For in vivo imaging applications, the soluble TCR willdesirably include a radionuclide (e.g., 1251, 32P, 99Tc) or otherdetectable tag which can be administered to a mammal and the subjectscanned by known procedures for binding of the TCR or fragment thereof.Such an analysis of the mammal could aid in the diagnosis and treatmentof a number of disorders including e.g. undesired expression of APCsaccompanying immune system disorders and cancer.

The invention can also be used for in vivo imaging of tumors bearingtumor-associated peptide antigens in a subject having or suspected ofhaving such a tumor. In the practice of this method, a composition isadministered to the subject that comprises a detectably labeled solubleTCR molecule or fragment thereof that specifically binds thetumor-associated peptide antigen in the context of a peptide/MHC complexon the tumor. The composition is administered in vivo for a period oftime sufficient to permit its accumulation at the tumor site. Theaccumulated composition is then detected so as to image the tumor.

The composition comprising the TCR can be administered parenterally(such as intravenously, intramuscularly, subcutaneously, intratumorally,etc.) at a locus and/or by a route providing access to the tissue, organor cells of interest. In other applications, the composition comprisingthe TCR can be administered intranasally, orally or transdermally.

The accumulated composition of the soluble TCR can be detected by avariety of means. These include detection by a detector selected fromthe group consisting of a conventional scintillation camera, a gammacamera, a rectilinear scanner, a PET scanner, a SPECT scanner, a MRIscanner, a NMR scanner, an ultrasound machine, an X-ray machine, aluminescence imaging system, and a fluorescence imaging system.

The imaging methods of the present invention further encompasspretargeting methods which in some applications may improve thedetection of a tumor cell or tissue. This approach uses a multi-stepprotocol. For example, a targeting TCR is conjugated with either avidinor biotin and then is administered, for example by injection, whereuponit localizes in the tumor of interest. Thereafter, either biotin oravidin (depending on which was coupled to the targeting antibody),bearing a label, is injected and becomes localized at the site of theprimary antibody by binding to avidin or biotin respectively.Alternatively other pairs of interacting molecules can substitute thebiotin/streptavidin molecules. Several pretargeting approaches have beendeveloped for antibodies (see Chang et al 2002. Mol. Cancer Therap.1:553-563) that could be used to pretarget TCR-based reagents.

The invention can also be employed in applications that involvefluorescence activated cell sorting (FACS). FACS can be used to detectinteractions between the soluble TCRs or fragments thereof and targetcells. For example, the soluble TCR can be biotinylated in accordancewith standard methods and combined with streptavidin-phycoerythrin (PE)to form labeled sc-TCR tetramers, for instance. However, as mentionedmultimerization will often not be needed. FACS can be used toqualitatively measure the interaction of the soluble TCR and a suitabletarget cell such as T2 cells and tumor cell lines.

The following Examples show the construction and characterization of anovel fusion protein comprising a soluble single chain HLA-A2.1restricted TCR that recognizes an unmutated p53 peptide spanning p53amino acid residues 264-272, genetically linked to human IL-2. Thepeptide specific binding of the TCR portion of the molecule to peptideloaded HLA-A2 as well as the specific IL-2 receptor binding capabilityand bioactivity of the IL-2 portion of the molecule was investigated.The Examples show that these types of TCR based fusion proteins canserve as an alternative to antibody based targeted tumor therapies or asan addition to other targeted tumor therapies such as antibody basedimmunocytokines. Separate and distinct approaches to targeting a tumormay demonstrate additive or synergistic antitumor effects.

The Examples further show construction and expression of a soluble threedomain mouse scTCR which recognizes human p53 peptide (aa 264-272) inthe context of HLA-A2.1. The three domain scTCR is fused to human IL-2yielding a soluble 264scTCR/IL-2 fusion protein which is expressed athigh levels and secreted from mammalian cells. The TCR portion of the264scTCR/IL-2 fusion protein retains its MHC-restricted, peptidespecific antigen binding properties, and the IL-2 portion binds to IL-2receptor and is biologically active. Moreover, the Examples further showthat this fusion protein is capable of conjugating target and effectorcells, exhibits favorable pharmacokinetics in mice, can bind to targettumor cells and has anti-tumor effects. Therefore, soluble scTCR fusionproteins will provide access to another repertoire of antigens fortargeted immunotherapy, which are not recognizable by antibody basedimmunotherapies. TCR-based therapies will serve as an alternative toantibody based treatments or as a useful addition to other targetedtumor therapies.

The present disclosure shows that soluble TCR has sufficient affinityfor peptide antigen to allow good detection. In particular, the affinityof the 264scTCR is sufficient to bind to unmanipulated tumor cells andeffectively conjugate target and effector cells.

A reported problem surrounding systemic administration of cytokines totreat tumors is the short serum half life and toxicity of theseproteins. Importantly, the 264scTCR/IL-2 fusion protein of the inventionhas an apparent serum half life of about 3 hours and appears to remainintact in the blood. Thus, the 264scTCR/IL-2 fusion protein effectivelyincreases the half life of IL-2 and survives intact in the blood,suggesting that it is a new agent for immunomodulatory cancer therapy.At higher doses than used in the Example, the serum half life of thepresent fusion protein should increase [3, 25, 37, 38], thereby furtherimproving the efficacy of the molecule against tumors.

There is recognition that IL-2 concentrated at the tumor site shouldactivate local T-cells as well as other IL-2 responsive cells, therebyrecruiting effector cells to the site of the tumor. Thus, byconcentrating IL-2 at the site of a tumor, the present TCR fusionmolecules may help potentiate a robust immune response includingactivation and proliferation of a variety of T-cell clones as well asactivation of NK cells or other members of the innate immune system.Such a multifaceted anti-tumor response will be more effective for theeradication of primary tumors as well as distant metastases.

The data show that it is possible to construct a biologically activebi-functional molecule comprised of a TCR and a cytokine. This fusionprotein is capable of binding to tumor cells, mediating the conjugationof target and effector cells, and has reasonable pharmacokineticproperties. Besides p53, other gene products that are upregulated andpresented in the context of MHC on tumor or virally infected cells maybe used as targets for the present TCR-based immunotherapies. Further,other immunomodulatory molecules such as GM-CSF, IFNγ, or IFN-α can belinked to the TCR to activate other effector cells for an anti-tumor oranti-viral response. These novel TCR fusions will form a new class ofimmunotherapeutics for the treatment of cancer and viral infection.

By the term “specific binding” or a similar term is meant a moleculedisclosed herein which binds another molecule, thereby forming aspecific binding pair. However, the molecule does not recognize or bindto other molecules as determined by, e.g., Western blotting ELISA, RIA,mobility shift assay, enzyme-immuno assay, competitive assays,saturation assays or other protein binding assays know in the art. Seegenerally, Ausubel, et al supra; Harlow and Lane in, Antibodies: ALaboratory Manual (1988) and references cited therein for examples ofmethods for detecting specific binding between molecules.

By the term “fully soluble” or similar term as it is meant to describe aTCR is meant that it is not readily sedimented under low G-forcecentrifugation from an aqueous buffer e.g., cell media. Further, asc-TCR fusion protein is soluble if the fusion protein remains inaqueous solution at a temperature greater than about 5-37° C. and at ornear neutral pH in the presence of low or no concentration of an anionicor non-ionic detergent. Under these conditions, a soluble protein willoften have a low sedimentation value e.g., less than about 10 to 50svedberg units. Aqueous solutions referenced herein typically have abuffering compound to establish pH, typically within a pH range of about5-9, and an ionic strength range between about 2 mM and 500 mM.Sometimes a protease inhibitor or mild non-ionic detergent is added anda carrier protein may be added if desired such as bovine serum albumin(BSA) to a few mg/ml. Exemplary aqueous buffers include standardphosphate buffered saline, Tris-buffered saline, or other known buffersand cell media formulations.

The following non-limiting examples are illustrative of the invention.

Example 1 Generation of TCR Fusion Protein Constructs

A fusion protein comprising a three domain, HLA-A2.1 restricted mouseTCR specific for a p53 peptide antigen fused to human IL-2 was made. Forthis TCR fusion protein construct, the Va and V0/C0 regions weregenerated by RT-PCR from RNA isolated from a mouse T cell clone thatproduces TCRs specific for human p53 (aa 264-272) peptide. Thecarboxyl-terminal end of the variable region of the TCRa chain (Va3) wasfused via a flexible linker (G₄S)₄ (SEQ ID NO: 17) [21] to theN-terminus of the V0 (V133) to generate the antigen binding portion ofthe TCR. The C0 domain, which is directly linked to the V0 domain, wastruncated at the amino acid residue just prior to the final cysteine,removing the transmembrane and cytoplasmic domains, to generate asoluble single-chain TCR molecule (FIGS. 1A and 1B). Human IL-2 wasfused to the TCR portion via a short linker (amino acid sequenceVNAKTTAPSVYPLAPV; SEQ ID NO:1). An EE tag (amino acid sequenceEEEEYMPME; SEQ ID NO:2) [11] was inserted just downstream of the IL-2portion of the fusion molecule to allow for detection of the TCR/IL-2fusion protein by an anti-EE tag mAb [11] if desired. Mammalian cellexpression is driven by a CMV promoter, secretion is directed by anantibody light chain leader, and selection was carried out by G418resistance.

FIG. 1 is explained in more detail as follows. 1A) Schematicrepresentation of the domain structure of the 264scTCR/IL-2 fusionprotein. 1B) Amino acid sequence of 264scTCR/IL-2 fusion protein Aminoacid numbers for each domain of the fusion protein are indicated in thefigure.

Example 2 Expression of TCR/IL-2 Fusion Protein in Mammalian Cells

To characterize the 264scTCR/IL-2 fusion protein, the 264scTCR/IL-2construct was stably transfected into CHO-K1 cells. Stable transfectantssecreting 264scTCR/IL-2 fusion protein were selected using ELISA assaysas described in Materials and Methods. Positive signals for these ELISAsindicate that the transfected cells secrete 264scTCR/IL-2 fusion proteinthat is recognized by both anti-murine TCR and anti-human IL-2antibodies suggesting that the secreted 264scTCR/IL-2 is properlyassembled and folded in the transfected cells and that it remains intactwhen it is secreted from the cells.

264scTCR/IL-2 fusion protein was purified from cell supernates byimmunoaffinity chromatography with a yield of approximately 1.8 mg/l ofsupernate. Purified fusion protein was subjected to SDS-PAGE andCoomassie staining. Under either reducing or non-reducing conditions,the predominant stained band migrated at approximately 60 kDa (FIG. 2),which is consistent with the predicted molecular mass for this proteinand indicates that the fusion protein remains intact with no unexpectedintramolecular disulfide bonds when it is secreted from the cells. Thelarger band in the nonreducing gel may be a dimer form of the fusionprotein. This conclusion is based on the observation that the largerband has the apparent molecular mass approximately twice that of thefusion protein and this band is reduced to the size of the fusionprotein under reducing conditions. The data indicate that thetransfected CHO cells produce 264scTCR/IL-2 fusion protein of theexpected molecular mass and that it is properly folded, assembled, andsecreted as a soluble fusion protein.

FIG. 2 is explained in more detail as follows. CHO cells were stablytransfected with the 264scTCR/IL-2 expression vector. The secretedfusion protein was purified by immunoaffinity chromatography andsubjected to SDS-PAGE under either reducing or non-reducing conditionsas indicated at the top of the figure. SDS-PAGE gels were stained withCoomassie brilliant blue.

Example 3 MHC/Peptide Binding Ability of the TCR Portion of the264scTCR/IL-2 Fusion Protein

The ability of the 264scTCR/IL-2 fusion protein to bind to peptideloaded MHC was determined by flow cytometry. T2 cells were loaded withp53 (aa 264-272) or p53 (aa 149-157) (control) peptide and stained with264scTCR/IL-2 fusion protein. Cells loaded with p53 (aa 264-272) stainedpositively with 264scTCR/IL-2 when detected with either the anti-TCR C13mAb or the anti-IL-2 detection antibody (FIGS. 3A and 3B). Cells loadedwith p53 (aa 149-157) control peptide did not stain with either theanti-TCR Cβ mAb or the anti-IL-2 detection antibodies. To demonstratethat the lack of staining of p53 (aa 149-157) loaded T2 cells is not dueto an inability of the p53 (aa 149-157) peptide to bind to HLA-A2, T2cells loaded with no peptide, p53 (149-157), or p53 (264-272) peptidewere stained with BB7.2 α-HLA-A2 monoclonal antibody. Cells loaded witheither p53 peptide stained stronger than cells loaded with no peptide,suggesting that both peptides are capable of binding to HLA-A2 molecules(FIG. 3C). T2 were also stained for IL-2 receptor and were found not toexpress IL-2 receptor; thus, these data indicate that binding of the264scTCR/IL-2 fusion protein is mediated by the TCR component of thefusion protein. The lack of staining by the fusion protein when T2 cellswere loaded with the control peptide also indicates that staining ismediated by the TCR component and that the staining is specific for theappropriate peptide. These data indicate that the TCR portion of the264scTCR/IL-2 fusion protein is capable of recognizing its specificpeptide in the context of HLA-A2.

FIG. 3 is explained more fully as follows. T2 cells were loaded with p53(aa 264-272) peptide (gray line) or p53 (aa 149-157) peptide (blackline), and stained with either 3A) 264scTCR/IL-2 fusion protein andanti-TCR Cβ mAb or 3B) 264scTCR/IL-2 fusion protein and anti-IL-2 mAb.3C): T2 cells loaded with p53 (aa 264-272) peptide (dark grey line), p53(aa 149-157) peptide (light grey line), or no peptide (black line) werestained with anti-HLA-A2 BB7.2 mAb followed by FITC labeled goatanti-mouse IgG. The shaded peak is unstained T2 cells.

Example 4 IL-2 Receptor Binding Ability of the IL-2 Portion of the264scTCR/IL-2 Fusion Protein

The IL-2 receptor binding capability of the IL-2 portion of the264scTCR/IL-2 fusion protein was studied by flow cytometry. Primarymouse splenocytes were isolated and stimulated with rIL-2 and anti-CD3to generate T cell blasts. Stimulated splenocytes that express IL-2receptor stained positively with p53 (aa 264-272) loaded HLA-A2tetramers only in the presence of 264scTCR/IL-2 fusion protein (FIG.4A). Likewise, CTLL-2 mouse cytotoxic T lymphocytes, whichconstitutively express IL-2 receptor, stained positively with the264scTCR/IL-2 fusion protein but not with a 264scTCR/kappa fusionprotein (FIG. 4B). When CTLL-2 cells were incubated with α-human CD25blocking antibody or isotype control antibody followed by 264scTCR/IL-2,staining was substantially reduced when the cells were incubated withthe blocking antibody but not with isotype control antibody. The lack ofsignal from the CTLL-2 cells incubated with a 264scTCR/mouse kappa chainfusion protein or with IL-2 receptor blocking antibody indicates thatstaining of these cells is mediated by the IL-2 portion of the264scTCR/IL-2 fusion protein. These data suggest that the IL-2 portionof the 264scTCR/IL-2 fusion protein is capable of binding to the IL-2receptor.

FIG. 4 is explained in more detail as follows. 4A): Mouse splenocyteswere stimulated with IL-2 and anti-CD3E mAb and then incubated in thepresence (gray line) or absence (black line) of 264scTCR/IL-2 fusionprotein. Bound fusion protein was detected with PE labeled HLA-A2 p53(aa 264-272) tetramers. 4B): CTLL-2 cells were incubated with α-humanCD25 blocking antibody or isotype control antibody followed by264scTCR/IL-2 or 264scTCR/kappa fusion protein. Bound fusion protein wasdetected with PE labeled α-TCR-V133 antibody. The shaded peak isunstained CTLL-2 cells. Black line: CTLL-2 cells stained with264scTCR/IL-2 only. Gray dotted line: CTLL-2 cells incubated withcontrol antibody followed by 264scTCR/IL-2. Light gray line: CTLL-2cells incubated with α-human CD25 blocking antibody followed by264scTCR/IL-2. Dark gray line: CTLL-2 cells stained with 264scTCR/kappafusion protein. Black dashed line: CTLL-2 cells stained with α-TCR-Vβ.

Example 5 Biological Activity of 264scTCR/IL-2 Fusion Protein

To demonstrate biological activity of the IL-2 portion of the264scTCR/IL-2 fusion protein, IL-2 dependent CTLL-2 cells were culturedwith either 264scTCR/IL-2 or recombinant IL-2 at various concentrationsand cell viability was assessed using WST-1. As shown in FIG. 5A, theability of rIL-2 or 264scTCR/IL-2 to support the growth of CTLL-2 cellswas dose dependent, wherein there was more cell proliferation at higherdoses of either recombinant IL-2 or 264scTCR/IL-2. Further, there weresimilar levels of cell proliferation when equivalent molar amounts ofeither recombinant IL-2 or 264scTCR/IL-2 were used. As a further controlfor specificity, CTLL-2 cells were incubated with 264scTCR/IL-2 withα-human CD25 blocking antibody or isotype control. When the blockingantibody was included in the culture, proliferation was substantiallydecreased with both concentrations of blocking antibody, butproliferation of the CTLL-2 cells was unaffected by either concentrationof control antibody (FIG. 5B). The data indicate that the IL-2 portionof 264scTCR/IL-2 has similar biological activity to recombinant IL-2 invitro and that the proliferation activity of the fusion protein isdependent on the IL-2 portion of the molecule.

The dissociation constant of the 264scTCR for its cognate MHC/peptidehas been found to be approximately 10⁻⁷ M at physiological conditionsusing surface plasmon resonance detection.

FIG. 5 is explained more fully as follows. 5A): CTLL-2 cells werecultured with 264scTCR/IL-2 (solid line) or recombinant IL-2 (dottedline) at various concentrations as indicated at the bottom of thefigure. 5B): CTLL-2 cells were incubated with 264scTCR/IL-2 and α-humanCD25 blocking antibody or isotype control antibody as indicated at thebottom of the figure. Cell viability was measured by incubation withWST-1 and absorbance was read at 450 nm Cab+S: 5 μg control antibody;Cab+50: 50 μg control antibody; Bab+S: 5 μg blocking antibody; Bab+50:50 μg blocking antibody.

Example 6 Conjugation of Cells Mediated by 264scTCR/IL-2 Fusion Protein

A useful property for the 264scTCR/IL-2 fusion protein would be capacityto bring together target and effector cells through its TCR and cytokineportions, respectively. To demonstrate that the 264scTCR/IL-2 fusionprotein can effectively conjugate cells, T2 cells were loaded witheither p53 (aa 264-272) or p53 (aa 149-157) peptides and then labeledwith dihydroethidium (HE). CTLL-2 cells were labeled with calcein AM andthe two labeled cell populations were mixed and incubated in thepresence or absence of 264scTCR/IL-2 fusion protein. Samples wereanalyzed by flow cytometry. When the two cell populations were incubatedin the absence of the 264scTCR/IL-2 fusion protein (FIGS. 6A and 6B) orwhen the T2 cells were loaded with control peptide and incubated withthe CTLL-2 cells in the presence of 264scTCR/IL-2 fusion protein (FIG.6C), the cells remained as two distinct populations on the flowcytometry histograms representing approximately 45% of the totalpopulation each (FIGS. 6A, 6B, and 6C, regions 1 and 3) with onlyapproximately 0.46% of the total population falling in the doublestained cell window (FIGS. 6A, 6B, and 6C, region 2). However, when theT2 cells were loaded with p53 (aa 264-272) peptide and incubated withthe CTLL-2 cells in the presence of the 264scTCR/IL-2 fusion protein(FIG. 6D), a double staining population of cells appears, representing4.1% of the total population (FIG. 6D, region 2, conjugated cells),suggesting that T2 cells were conjugated to CTLL-2 cells via the264scTCR/IL-2 fusion protein.

FIG. 6 is explained in more detail as follows. T2 cells were loaded witheither p53 (aa 264-272) (6B and 6D) or p53 (aa 149-157) (control)peptides (6A and 6C) and then labeled with HE. CTLL-2 cells were labeledwith calcein AM. Labeled cells were mixed and incubated in the presence(6C and 6D) or absence (6A and 6B) of 264scTCR/IL-2 fusion protein andthe samples were analyzed by flow cytometry. Assay conditions includingloading peptide used, and presence or absence of fusion protein, areindicated beneath each histogram. Single stained regions are marked 1and 3 and the double stained cell population is marked 2.

Example 7 Pharmacokinetics of 264scTCR/IL-2 in Mice

The pharmacokinetics of the 264scTCR/IL-2 fusion protein was measured inBALB/c mice. Mice were injected intravenously and serum samples werecollected various time points. The serum levels of 264scTCR/IL-2 fusionprotein were measured using ELISA. The ELISA detection was performedusing anti-TCR mAb capture/anti-IL-2 Ab detection (FIG. 7A), anti-TCRmAb capture/anti-TCR mAb detection (FIG. 7B), or anti-IL-2 mAbcapture/anti-IL-2 polyclonal Ab detection (FIG. 7C) to determine whetherthe fusion protein is modified or cleaved in vivo. Mice injected with264scTCR/IL-2 fusion protein showed no obvious signs of toxicity. Inthese assays a maximum concentration of 0.75 to 2.5 μg/ml of264scTCR/IL-2 was detected with an apparent serum half-life of 1.6 to3.0 hours depending on the ELISA format used (FIG. 7). Since thereported serum half-life of free IL-2 is only about 5 minutes [6], thesedata indicate that the fusion protein is not cleaved in vivo but insteadremains intact for a relatively long period of time in the blood. Thesmall variability in the half-life of 264scTCR/IL-2 determined in thesestudies is most likely due to the differences in the sensitivity of theELISA assays.

FIG. 7 is explained in more detail as follows. BALB/c mice were injectedwith 264scTCR/IL-2 fusion protein and serum samples were collected at 15and 30 minutes, 1, 4, 8, and 24 hours post injection. Serumconcentrations of 264scTCR/IL-2 were determined by ELISA using thefollowing formats: 7A): Anti-TCR mAb capture/anti-IL-2 Ab detection;7B): Anti-TCR mAb capture/anti-TCR mAb detection; and 7C): Anti-IL-2 mAbcapture/anti-IL-2 Ab detection.

Example 8 Tumor Cell Staining with 264scTCR/IL-2

It would be useful if the 264scTCR/IL-2 fusion protein could recognizeand bind to its target tumor cells. To test whether the 264scTCR/IL-2 iscapable of binding to tumor cells, A375 human melanoma cells, whichexpress both HLA-A2.1 and p53, were stained with either 264scTCR/IL-2 or3C8, an irrelevant TCR/IL-2 fusion protein. Cells not incubated withfusion protein and cells incubated with 3C8 did not stain with theH57-597 detection antibody, while cells incubated with 264scTCR/IL-2stained positively with the detection antibody (FIG. 8). This resultsuggests that the 264scTCR/IL-2 fusion protein is capable of recognizingand binding to its target tumor cell and is useful as an anti-cancertherapeutic in vivo.

FIG. 8 is explained more fully as follows. A375 human melanoma cellswere incubated with no fusion protein (dashed black line), 5 μg 3C8TCR/IL-2 fusion protein (control) (dotted line), or 5 μg 264scTCR/IL-2fusion protein (solid black line) followed by staining with H57-597 mAb.Unstained cells are represented by the shaded area.

Example 9 Anti-Tumor Effects of 264scTCR/IL-2 Fusion Protein

To determine if the 264scTCR/IL-2 fusion protein has anti-tumor activityin vivo, an experimental metastasis assay was performed. Female athymicnude mice were injected with the highly metastatic A375 human melanomasubclone, A375-C15N, and treated with varying doses of either264scTCR/IL-2 or recombinant IL-2. Forty-two days after tumor cellinjection, lung nodules were counted. Both 264scTCR/IL-2 and recombinantIL-2 reduced lung metastasis in a dose dependent manner (FIG. 9).However, at all doses lung metastasis was reduced to a greater degreewith the 264scTCR/IL-2 fusion protein, suggesting that targeting thecytokine to the tumor may provide greater efficacy as a cancertherapeutic.

Mice treated with either 264scTCR/IL-2 or recombinant IL-2 showed noobvious signs of toxicity. Both treatments resulted in reduction of lungmetastasis; however, at all doses treatment with 264scTCR/IL-2 was moreeffective than recombinant IL-2.

FIG. 9 is explained more fully as follows. Female athymic nude mice wereinjected with highly metastatic A375-C15N cells and treated with264scTCR/IL-2, recombinant IL-2, or PBS. Forty-two days after tumor cellinjection, the lungs were removed, lung nodules were counted, and themean number of lung nodules relative to the PBS treated control groupwas plotted.

Example 10 Flow Cytometric Analysis of Staining of Peptide-Loaded T2Cells by Monomeric and Multimeric 264scTCR Fusion Proteins

Monomeric or multimeric forms of various 264scTCR fusion proteins wereprepared and their binding to T2 cells was analyzed by flow cytometry asdescribed in Methods, sections 11 and 12, below. The results, shown inFIG. 10, demonstrate that the 264scTCR fusion proteins stained p53(aa264-273)-loaded T2 cells (FIG. 10B) to a greater degree than p53(aa149-157)-loaded cells (FIG. 10A). In the figures, unstained T2 cellsare shown in the histogram labeled T2 149 unstained.001; p53(aa149-157)- and p53 (aa264-273)-loaded T2 cells stained with thesecondary reagent (H57-PE) are shown in the histograms labeled “T2 149H57.002” and “T2 264 H57.009”, respectively; p53 (aa149-157)- and p53(aa264-273)-loaded T2 cells stained with multimeric 264scTCR/IgG1followed by H57-PE are shown in the histograms labeled “T2 149 IgGH57.003” and “T2 264 IgG H57.010”, respectively; p53 (aa149-157)- andp53 (aa264-273)-loaded T2 cells stained with 264scTCR/IL-2 followed byH57-PE are shown in the histograms labeled “T2 149 IL2 H57.004” and “T2264 IL2 H57.011”, respectively; p53 (aa149-157)- and p53(aa264-273)-loaded T2 cells stained with monomeric 264scTCR/trunIgG1followed by H57-PE are shown in the histograms labeled “T2 149 trunH57.005” and “T2 264 trun H57.012”, respectively; and p53 (aa149-157)-and p53 (aa264-273)-loaded T2 cells stained with monomeric 264scTCR/BirAfollowed by H57-PE are shown in the histograms labeled “T2 149 birAH57.006” and “T2 264 birA H57.013”, respectively. This result confirmedthat the observed staining is peptide-specific.

Monomeric forms of the 264scTCR were able to stain to some degree. Forexample, the mean channel fluorescence (MCF) for staining with the264scTCR/trunIgG form increased from 10.95 for the p53(aa149-157)-loaded cells to 55.34 for the p53 (aa264-273)-loaded cells.Similarly, the MCF for the 264scTCR/BirA form increased from 13.41 forp53 (aa149-157)-loaded cells to 95.14 for the p53 (aa264-273)-loadedcells. Multimeric forms of the 264scTCR were able to specifically stainthe peptide-loaded T2 cells to an even greater extent. For example, theMCF for the 264scTCR/IgG1 form increased from 119 for p53(aa149-157)-loaded cells to 863 for the p53 (aa264-273)-loaded cells.

Example 11 Staining of Tumor Cells by 264scTCR Fusion Proteins

The ability of the 264scTCR reagents to stain tumor cells was alsotested. Cultured A375 cells were detached with 10 mM EDTA in PBS (pH7.4)and washed twice with washing buffer. Cell staining was carried outusing 4 μg 264scTCR/IgG1 fusion protein for 45 minutes at 23° C. Thecells were washed once and stained with 3 μg FITC-conjugated F(ab′)₂fragment of goat anti-human IgG Fc (anti IgG-FITC). After washing twice,the stained cells were resuspended and analyzed on a FACScan. A375 cellsstained with anti IgG-FITC alone were run as a control.

Referring to FIG. 11A, the results of this analysis show that A375 tumorcells could be stained with the 264scTCR/IgG1 fusion protein. In thispanel (11A), A375 cells stained with anti IgG-FITC alone or264scTCR/IgG1 followed by anti IgG-FITC are shown in histograms labeled“A375-FITC.005” and “A375-264.FITC.006”, respectively. Additionalexperiments using A375 tumor cells were performed to furthercharacterize optimal staining conditions. For example, PE-conjugatedanti-human IgG antibody (anti IgG-PE) (FIG. 11B) or PE-conjugated H57mAb (FIG. 11D) were used in place of the FITC-conjugated antibody as asecondary reagent. In FIG. 11B, A375 cells stained with anti IgG-PEalone or 264scTCR/IgG1 followed by anti IgG-PE are shown in histogramslabeled “A375-PE.007” and “A375-264.PE.008”, respectively. In FIG. 11D,A375 cells stained with H57-PE alone or 264scTCR/IgG1 followed by H57-PEare shown in histograms labeled “A375-H57PE.009” and“A375-264.H57PE.010”, respectively. In each case, the 264scTCR/IgG1stained the A375 tumor cells. Biotinylated 264scTCR/BirA that had beenmultimerized with streptavidin-PE (SA-PE) was also used to stain A375cells (FIG. 11C), and showed increased staining compared with the cellsstained with streptavidin-PE alone. Referring to FIG. 11C, A375 cellsstained with SA-PE alone or biotinylated 264scTCR/BirA complexed withSA-PE are shown in histograms labeled “A375-SAPE.001” and“A375-264BtnSaPE.002”, respectively.

Example 12 Staining of Fixed Cells by 264scTCR Fusion Proteins Detectedby Flow Cytometry

As discussed, the ability to detect MHC/peptide complexes in preservedor “fixed” samples is advantageous, especially in clinical or othermedical settings where it is typical practice to fix cells, tissues orother biological samples taken from patients. However, since theMHC/peptide complex represents a cell surface antigen composed of threeseparate polypeptide chains, it is uncertain that the structuralintegrity of the MHC/peptide complex would remain sufficiently intactfor detection by the soluble TCR following typical fixation procedures.To assess whether soluble TCR staining could be carried out on fixedcells, peptide-loaded T2 cells and unmanipulated A375 tumor cells wereanalyzed by flow cytometry. Cultured A375 cells were detached with 10 mMEDTA in PBS (pH 7.4) and washed twice with washing buffer. T2 cells wereincubated with 50 μM p53 (aa264-273) for 3 hours and then washed twicewith washing buffer. Both cell types were fixed with 3.7% formaldehydefor 5 minutes and washed twice. Cell staining was carried out using 4 μg264scTCR/IgG1 or CMVscTCR/IgG1 fusion protein in the presence or absenceof 20 μg HLA-A2.1/p53 (aa 264-272) tetramers for 45 minutes at 23° C.The cells were washed once and stained with 3 μg FITC-conjugated F(ab′)₂fragment of goat anti-human IgG Fc. After washing twice, the stainedcells were resuspended and analyzed on a FACScan.

Referring to FIG. 12A, the results showed that the 264scTCR/IgG1 fusionprotein positively stained the formaldehyde-fixed A375 cells (histogramlabeled “A375F-264.006”), while staining with CMVscTCR/IgG1 (histogramlabeled “A375F-CMV.005”) was not detectable above background. Since theCMV peptide is not present on A375 cells, use of the CMVscTCR/IgG1control reagent provides a measure of any non-specific interactionsbetween the TCR or IgG1 domains with the tumor cells. By determining thedifference in tumor cell staining between the 264scTCR/IgG1 fusionprotein and the CMVscTCR/IgG1 control, this method allows directmeasurement of the level of tumor antigen presentation on the surface ofthe fixed tumor cell sample.

To confirm that A375 cell staining with 264scTCR/IgG1 fusion protein wasTCR-specific, HLA-A2.1/p53 (aa 264-272) tetramers were used as blockingreagents. Staining of A375 cells with 264scTCR/IgG1 was reduced by theaddition of HLA-A2.1/p53 (aa 264-272) tetramer blocking reagent(histogram labeled “A375F-264TET.264.008”), further indicating that264scTCR/IgG1 can specifically bind to tumor cells. As expected,addition of HLA-A2.1/pCMV tetramer reagent to A375 cells stained with264scTCR/IgG1 did not have any effect on the specific staining of the264scTCR/IgG1 reagent (histogram labeled “A375F-264TET.CMV.007”).Similar results were seen with the peptide-loaded T2 cells (FIG. 12B).

These results demonstrate the monomeric and multimeric soluble TCRreagents can specifically stain cells presenting a peptide in thecontext of an MHC complex. Furthermore, soluble TCR reagents canspecifically stain unfixed and fixed tumor cells presenting a tumorantigen in context of an MHC complex. In addition, specific staining ofthe cells by the soluble TCR reagent could be reduced by the addition ofa completing MHC molecule that binds the soluble TCR reagent. Additionof a 1 to 100 fold molar excess of the completing MHC molecule over thesoluble TCR reagent in control staining reactions is particularly usefulin distinguishing the specific binding component (i.e. binding topeptide-MHC) versus the non-specific binding component of soluble TCRstaining. This is relevant when comparing staining of different cellsand tissues that may exhibit different degrees of non-specific andspecific soluble TCR binding. For example, variability between thenon-specific binding of the soluble TCR in different samples (cells ortissues) would make it extremely difficult to determine the degree ofspecific soluble TCR binding without the use of an appropriatecompleting MHC molecule in a control staining reaction.

Example 13 Staining of Fixed Cells by 264scTCR Fusion Proteins Detectedby Immunofluorescent Microscopy

Several cell lines that vary with respect to HLA-A2 and p53 expression(i.e., A375, HT29 and Saos2) were chosen for analysis and stained witheither 264scTCR/IgG1 fusion protein or control fusion proteinCMVscTCR/IgG1. The cells were cultured on cover slips for 24 hours andthen fixed with 3.7% formaldehyde for 5 minutes and washed twice withwashing buffer (0.5% BSA and 0.1% sodium azide in PBS). The BSArepresents a blocking agent to reduce nonspecific protein binding. Thecells were stained with 10 μg 264scTCR/IgG1 or CMVscTCR/IgG1 fusionprotein in 200 μl of PBS containing 5% normal goat serum (NGS) for 45minutes at 23° C. The NGS represents a blocking agent to reducenonspecific binding. The cells were washed twice and stained with 3 μgFITC-conjugated F(ab′)₂ fragment of goat anti-human IgG Fc□ (JacksonImmunoResearch, West Grove, Pa.). The cells were washed twice and thenonce with equilibration buffer (Molecular Probes, Eugene, Oreg.). Coverslips were mounted on glass slides with anti-fade reagent in glycerolbuffer (Molecular Probes, Eugene, Oreg.) and sealed with nail oil. Theslides were documented using a Nikon epi-fluorescence microscope (Nikon,Tokyo, Japan) with a SPOT RT camera and SPOT RT software v3.2(Diagnostic Instrument, Sterling Heights, Mich.).

For HLA-A2 staining, the fixed cells were stained with 10 μg BB7.2, amouse anti-human HLA-A2 antibody, in 200 μl of PBS containing 5% normalgoat serum (NGS) for 45 minutes at 23° C. The cells were washed twiceand stained with 4 μg FITC-conjugated F(ab′)₂ fragment of goatanti-mouse IgG Fc□ (Jackson ImmunoResearch, West Grove, Pa.). The cellswere washed twice and then once with equilibration buffer (MolecularProbes, Eugene, Oreg.). Cover slips were mounted and documented asdescribed above.

For p53 staining, the fixed cells were permeablized with 0.2%TrintonX-100 for 20 minutes and then stained with 10 μg PAb122, a mouseanti-p53 antibody, in 200 μl of PBS containing 5% normal goat serum(NGS) for 45 minutes at 23° C. The cells were washed twice and stainedwith 4 μg FITC-conjugated F(ab′)₂ fragment of goat anti-mouse IgG Fc□(Jackson ImmunoResearch, West Grove, Pa.). The cells were washed twiceand then once with equilibration buffer (Molecular Probe, Eugene,Oreg.). Cover slips were mounted and documented as described above.

As shown in FIG. 13, A375 cells stained positively for HLA-A2 and p53,HT29 stained positively for p53 but not HLA-A2 and Saos2 cells stainedpositively for HLA-A2 but not p53 Immunofluorescent staining with264scTCR/IgG1 was only detected for the A375 cells and none of the cellsstained positively with the CMVscTCR/IgG1. These results confirm thatthe presence of the HLA-A2 and the p53 antigen are required for positivestaining with the 264scTCR reagents. No background staining was seenwith the non-specific CMVscTCR reagent in any of the tumor cell lines orwith the 264scTCR reagent when the HLA-A2 and p53 antigen were notexpressed.

Example 14 Quantitative Staining Using 264scTCR Fusion Proteins

The number of 264scTCR complexes capable of binding peptide-loaded T2cells was determined T2 cells were incubated with various amounts of p53(aa264-273) for 3 hours and then washed twice with washing buffer. Cellstaining was carried out using 3.7 μg 264scTCR/BirA-streptavidin-PEtetramers for 45 minutes at 23° C. After washing twice, the stainedcells were resuspended and analyzed on a FACScan. Alternatively, cellstaining was carried out using 3.76 μg 264scTCR/IgG1 fusion protein for45 minutes at 23° C. The cells were washed once and stained with 3 μgPE-conjugated anti-human IgG antibody. After washing twice, the stainedcells were resuspended and analyzed on a FACScan.

The results of this analysis are shown in FIG. 14 for 264scTCR/BirAtetramers, and in FIG. 15 for 264scTCR/IgG1 fusions. Increasing level ofstaining with increasing amount of p53 peptide was observed for both the264scTCR/BirA tetramers and the 264scTCR/IgG1 fusions. To quantitate thenumber of complexes staining the cells, the level of fluorescenceintensity on stained cells was compared with the fluorescenceintensities of calibration beads having known numbers of PE moleculesper bead (QuantiBRITE PE beads; BD Biosciences), thus providing a meansof quantifying PE-stained cells using a flow cytometer.

The calculated number of complexes/cell with various concentrations ofpeptide for the 264scTCR/BirA tetramers and the 264scTCR/IgG1 fusionsare plotted in FIG. 16. The results show that the binding of as few as400 scTCR complexes could be detected on the stained cells. In addition,staining with the 264scTCR/IgG1 fusion followed by PE-conjugatedanti-human IgG antibody gave about a 4-10 fold increase in stainingcompared to that seen with the 264scTCR/BirA tetramers. This increase ispossibly the result of a higher level of PE conjugation to the antibodyand/or multiple antibodies reacting with the same 264scTCR/IgG1 fusion.

From the foregoing it will be appreciated that methods such as thosedescribed above to quantitatively detect TCR binding will be beneficialin optimizing the detection of rare antigens. The methods were appliedto detect 264scTCR reagent binding to tumor cells. Cells were preparedas described and stained with various amounts of264scTCR/BirA-streptavidin-PE tetramers for 45 minutes at 23° C. Afterwashing twice, the stained cells were resuspended and analyzed on aFACScan. Alternatively, cell staining was carried out with variousamounts of 264scTCR/IgG1 fusion protein for 45 minutes at 23° C. Thecells were washed once and stained with 2.5 μg PE-conjugated H57antibody. After washing twice, the stained cells were resuspended andanalyzed on a FACScan.

In each case, the number of complexes staining the cells was determinedby comparing the level of fluorescence intensity on stained cells withthe fluorescence intensities of calibration beads with known numbers ofPE molecules per bead. FIGS. 17A and 17B show staining of A375 tumorcells with the increasing amounts of 264scTCR reagents. FIGS. 18 and 19respectively show the quantitation of the staining observed for threetumor cell lines (A375, HT29 and Saos2) with the increasing amounts of264scTCR/BirA and 264scTCR/IgG1 reagents. The HLA-A2/p53 positive A375tumor cell line stained with both reagents and bound 2-5 fold more264scTCR reagent than the HT29 (HLA-A2-negative) and Saos2(p53-negative) cell lines. In addition, specific staining of the A375cells increased as the amount of 264scTCR reagent was increased.Differential detection of as few as 500 staining complexes could bedetermined by comparing the staining of the A375 cells with that ofother tumor cell lines. The results of these quantitative stainingstudies indicate that specific binding of the 264scTCR reagents to asfew as 300-500 HLA-A2/peptide complexes per cell can be readilydetected. In addition, the sensitivity of these staining reactions couldbe increased and optimized with the use of different TCR and secondaryreagents.

Example 15 Immunohistochemical Staining of Unmanipulated Tumor Tissue by264scTCR Fusion Proteins

To produce subcutaneous tumors, A375 human melanoma cells (1×10⁶) wereinjected subcutaneously into the left shoulder of nude mice. Tumors wereallowed to grow to 500 mm³ and the mice were humanely sacrificed. Tumorswere excised with overlying skin and fixed overnight in neutral bufferedformalin. For production of metastatic lung nodules, MDA-MB-231 cells(1×10⁶) were injected into the lateral tail vein of nude mice, andmetastatic lung nodules were allowed to develop. After 18 days, micewere humanely sacrificed, and the lungs were removed and fixed inneutral buffered formalin. Fixed tissue was dehydrated by sequential 30minute incubations in 70%, 90%, 95%, 100% (twice) ethanol followed bytwo 30 minute incubations in xylene. Tissues were then embedded inparaffin and 5 μm sections were prepared and mounted on microscopeslides.

For immunohistochemical staining, sections were rinsed twice for fiveminutes each in xylene followed by rehydration in sequential incubationsin 100% (twice), 95%, and 85% ethanol for two minutes each. After two 5minute washes with PBS and one 5 minute wash with the distilled water,slides were incubated in 3% H₂O₂ for 5 minutes to deactivate endogenousperoxidases followed by one five minute wash in the distilled water.Slides were placed in antigen retrieval solution (Dako) and heated to97° C. for 20 minutes. The slides were allowed to cool in the antigenretrieval solution at room temperature for 20 minutes followed by twofive minute washes in PBS.

If using a non-HRP labeled secondary reagent, slides were incubated inavidin/biotin blocking solution (ten minutes in each solution) followedby two five-minute washes in PBS. Slides were blocked in 1% normal goatserum (NGS) in PBS for 30 minutes at room temperature. This blockingstep is necessary to reduce background staining due to non-specificinteraction of the secondary goat antibody reagent. The slides were thenincubated for 45 minutes at room temperature in the presence or absenceof 10 μg (per 100 μl in 1% NGS) 264scTCR/IgG1 fusion protein or controlCMVscTCR/IgG1 fusion protein. After two five minute washes in PBS,slides were incubated in 1.6 μg (per 200 μl 1% NGS) HRP-labeled F(ab′)₂fragment of goat anti-human IgG Fcγ for 45 minutes at room temperature.Slides were washed twice for five minutes each with PBS. Slides wereincubated in DAB solution (Dako) until a light background appeared.Slides were rinsed in tap water and counterstained in hematoxylin for 15seconds. After washing with tap water, slides were rinsed in three bathsof 100% ethanol and three baths of xylene for 3 minutes each and thenmounted with Permount (Fisher). The level of tissue staining wasassessed by light microscropy and documented with a SPOT RT camera andSPOT RT software v3.2 (Diagnostic Instrument, Sterling Heights, Mich.).

A typical immunohistochemical analysis using A375 tumor sections isshown in FIGS. 20 and 21. The results showed that A375 tissue sectionsstained much more intensely (i.e., appeared more darkly colored) whenincubated with 264scTCR/IgG1 fusion protein compared to theCMVscTCR/IgG1 fusion protein or the secondary antibody alone. Thebackground staining observed with the CMVscTCR/IgG1 fusion protein iscomparable to that seen following incubation with a human IgG1 antibodyfollowed by HRP-labeled anti-human IgG antibody, indicating that thebackground staining is likely due to interaction of the IgG1 domain withthe tissue sections. In addition, staining of the mouse stromal tissueby the 264scTCR/IgG1 fusion protein was considerably less than that seenin the A375 tumor tissue present in the same section. These resultsindicate that the 264scTCR reagent is capable of specifically stainingfixed human tumor tissue sections by immunohistochemical methodstypically used to characterize human tumor samples.

Example 16 Immunohistochemical Staining of Tumor Histoarrays with264scTCR Fusion Proteins

Human tumor histoarrays are obtained from commercial sources or from theTissue Array Research Program (NCI). For staining, the histoarray slidesare rinsed twice for five minutes each in xylene followed by rehydrationin sequential incubations in 100% (twice), 95%, and 85% ethanol for twominutes each. After two 5 minute washes with PBS and one 5 minute washwith the distilled water, slides are incubated in 3% H₂O₂ for 5 minutesto deactivate endogenous peroxidases, followed by one five minute washin the distilled water. Slides are placed in antigen retrieval solution(Dako) and heated to 97° C. for 20 minutes. The slides are allowed tocool in the antigen retrieval solution for 20 minutes followed by twofive-minute washes in PBS. If using a non-HRP labeled secondary reagent,slides are incubated in avidin/biotin blocking solution (ten minutes ineach solution) followed by two five minute washes in PBS. Slides areblocked in 1% normal goat serum (NGS) in PBS for 30 minutes at roomtemperature and then incubated for 45 minutes at room temperature in thepresence or absence of 264scTCR/IgG1 fusion protein or CMVscTCR/IgG1fusion protein (or other non-binding scTCR reagent). After two fiveminute washes in PBS, slides are incubated in secondary reagent (eitherHRP-labeled goat anti-human IgG or biotinylated anti-TCR Cβ antibody)for 45 minutes at room temperature. Slides are washed twice for fiveminutes each with PBS.

If a non-HRP secondary reagent is used, slides are incubated withstreptavidin peroxidase solution for 15 minutes at room temperaturefollowed by two five minute washes with PBS. Alternatively,scTCR/BirA-streptavidin peroxidase reagents are used as stainingreagents in place of the reagents described above.

Slides are incubated in DAB solution (Dako) until a light backgroundappears. Slides are rinsed in tap water and counterstained withhematoxylin for 15 seconds. After washing with tap water, slides arerinsed in three changes of 100% ethanol and three changes of xylene andthen mounted with Permount (Fisher). The level of tissue staining isassessed by light microscropy and photographed, for example with a SPOTRT camera and SPOT RT software v3.2 (Diagnostic Instrument, SterlingHeights, Mich.).

Tumors that express HLA-A2 and p53 are expected to be differentiallystained when incubated with the 264scTCR fusion protein compared to theCMVscTCR fusion protein. Little or no staining is expected when thehistoarrays are incubated with no fusion protein. In addition, tumortissues that are negative for HLA-A2 and/or p53 are expected to showreduced staining with 264scTCR fusion protein compared toHLA-A2/p53-positive tumor tissue. This can give useful information aboutwhat types of tumors, and the relative proportions thereof, that can berecognized by 264scTCR fusion proteins, aiding decisions about theadvisability of treating a given type of tumor with a 264scTCR basedtherapy.

Example 17 Imaging of Tumors In Vivo with Fluorescent TCR Reagents

Expression vectors are constructed to generate 264scTCR fused to GFP(green fluorescent protein) or Luc (firefly luciferase). These vectorscan be generated from the 264scTCR/IgG1 expression vector describedherein by replacing the IgG1 gene fragment with GFP or Luc codingsequences. Sources of these coding sequences are commercially available(for example, pEGFP-C1 (Clontech) for the GFP gene, and pSP-Luc(Promega) for the Luc gene. The vectors are used as a template toisolate the appropriate DNA sequence by standard PCR methods. Expressionvectors for control TCR (i.e., CMVscTCR) fusions to GFP and Luc can begenerated by the same methods. In some applications these expressionvectors can be used to transfect cells such as CHO cells and theresulting expressed proteins are purified as described herein.

These purified proteins are used to image tumors in vivo. Human tumorcells that vary with respect to HLA-A2 and p53 expression are implantedeither subcutaneously or intravenously and tumors or metastatic lungnodules are allowed to develop as described in Example 15 above. For thescTCR/Luc fusions, mice are injected intravenously with increasingamounts of the scTCR/Luc fusion proteins. After a period of timenecessary to allow the fusion proteins to circulate throughout the body,the mice are injected intraperitoneally with 2.0 mg D-luciferinsubstrate for luciferase in 100 μl PBS, then anesthetized with xylazine(3 mg/ml) and ketamine (7 mg/ml) in PBS at 120 μl/20 g body weight. Forthe scTCR/GFP fusions, mice are injected intravenously with increasingamounts of the scTCR/Luc fusion proteins. After a period of timenecessary to allow the fusion proteins to circulate throughout the body,the mice anesthetized with xylazine (3 mg/ml) and ketamine (7 mg/ml) inPBS as described above.

For tumor detection in vivo, anesthetized mice are placed inside aNightOwl LB 981 Molecular Light Imager. Imaging is performed using atwo-step process and WinLight software (Berthold Technologies, OakRidgeTN). First, a black and white photographic image is acquired usinga 15 ms exposure followed by luminescent image acquisition using a5-minute photon integration period with background subtraction. Theluminescent image is processed in the software to colorize theluminescence intensity and then overlaid onto the black and whitephotographic image for presentation. In some cases mice are sacrificedand pathological assessment is performed to determine the size, locationand nature (i.e., antigen positivity or negativity) of the tumors.

Results from imaging studies demonstrating differential detection of the264scTCR/Luc or 264scTCR/GFP reagents at tumor sites bearing HLA-A2/p53positive tumor cells compared with other tissues indicate scTCR reagentscapable of specifically detecting tumors in vivo.

Additionally, results demonstrating differential detection of the264scTCR/Luc or 264scTCR/GFP reagents at tumor sites bearing HLA-A2/p53positive tumor cells compared with that of the CMVscTCR/Luc orCMVscTCR/GFP (control) reagents would further indicate those scTCRreagents capable of specifically detecting tumors in vivo. Imagingresults demonstrating differential detection of the 264scTCR/Luc or264scTCR/GFP reagents at tumor sites bearing HLA-A2/p53 positive tumorcells compared with results for tumor sites bearing HLA-A2-negative orp53-negative tumor cells further indicate those scTCR reagents arecapable of specifically detecting tumors in vivo.

Example 18 Imaging of Tumors In Vivo with Radiolabeled TCR

In another embodiment, 264scTCR fusion proteins are radiolabeled, forexample by direct iodination with ¹³¹I. Iodination is carried out usingstandard methods. Human tumor cells that vary with respect to HLA-A2 andp53 expression are implanted either subcutaneously or intravenously andtumors or metastatic lung nodules are allowed to develop as described.Mice are injected intravenously or intraperitoneally with radiolabeled264scTCR fusion protein and imaged for example at 1, 2, 4, 8, and 12hours and 1 to 14 days after injection of radiolabeled 264scTCR fusionprotein. For whole body scans, mice are anesthetized with 100 mg/kgsodium pentobarbital and imaged for example with a large field-of-viewSopha DSX camera fitted with a 4 mm pinhole collimator interfaced to amicrocomputer. Results from imaging studies demonstrating differentialdetection of the radionucleotide-labeled 264scTCR reagents at tumorsites compared with other tissue would indicate those radiolabelledscTCR reagents useful for specifically detecting tumors in vivo.

The following materials and methods were used as needed to conductexperiments outlined in the Examples.

1. Materials

A2.1 264 CTL clone #5 was derived by limiting dilution cloning [50] froma CTL line specific for the human p53 264-272 peptide generated inHLA-A2.1 transgenic mice [49]. CHO.K1 Chinese hamster ovary, Jurkathuman T lymphocyte, CTLL-2 mouse cytotoxic T lymphocyte, T2 humanlymphoblast, A375 human melanoma, H57-597 hybridoma, and BB7.2 hybridomacell lines were obtained from American Type Culture Collection(Rockville, Md.). The T2 human lymphblast cells are positive forHLA-A2.1 but deficient in TAP 1 and 2 proteins, which allows them todisplay empty MHC molecules that can then be loaded with exogenouspeptide [2]. The A375 human melanoma cell line was tested in ourlaboratory for both HLA-A2.1 and p53 and was found to be positive forboth antigens. The H57-597 hybridoma produces a monoclonal antibody thatrecognizes an epitope in the murine TCR β constant region, and the BB7.2hybridoma produces the BB7.2 monoclonal antibody that specificallyrecognizes an epitope on the alpha 2 domain of HLA-A2. The highlymetastatic subclone of the human melanoma cell line A375, A375-C15N,which was used only for in vivo metastasis studies was maintained aspreviously reported [53]. Recombinant human IL-2 and biotinylatedanti-human IL-2 polyclonal antibodies used for the ELISA in thepharmacokinetic study were purchased from R&D Systems, Inc.(Minneapolis, Minn.). Anti-TCR C13 mAb H57-597, anti-murine TCR Vβ3 mAb,anti-murine CD3ε mAb, anti-human IL-2 mAb, anti-human CD25 blockingantibody and isotype control antibody, and FITC labeled goat anti-mouseIgG were obtained from Pharmingen (San Diego, Calif.). All cell culturemedia and additives were purchased from CellGro (Herndon, Va.), and allcell culture materials were purchased from Nunc (Rochester, N.Y.) unlessotherwise noted. All mice were purchased from Harlan Labs (Indianapolis,Ind.).

2. Cell Culture

All cell lines were maintained in complete culture medium comprised ofIMDM supplemented with 10% heat inactivated FBS, 2 mM L-glutamine, and 1mg/ml G418 (for transfected CHO cells only) at 37° C. and 5% CO₂. CTLL-2cells were maintained in the same medium with the addition of 9 U/mlrecombinant human IL-2. A375-C15N cells were maintained in RPMI-1640with 10% heat inactivated FBS, penicillin and streptomycin (LifeTechnologies).

Mouse splenocytes were isolated by pressing spleens asepticallydissected from BALB/c mice through a nylon mesh screen and washing withculture medium. Red blood cells were lysed with Gey's solution for 2minutes followed by addition of culture medium to stop the lysis. Singlecell pellets were washed twice, resuspended at 2.5×10⁶ cells per mL inculture medium and cultured in complete culture medium containing 50 μM2-ME, 100 IU/mL recombinant human IL-2, and 50 ng/ml anti-murine CD3EmAb.

3. Constructs Primers—

Oligonucleotide primers were synthesized from sequences matching orcomplementing the mouse T cell receptor and human IL-2 genes:

KC228: (SEQ ID NO: 3) 5′-GAGGTGGCCCAGCCGGCCATGGCCCAGTCAGTGACGCAGC-3′;KC229: (SEQ ID NO: 4) 5′-GAGGTGACTAGTGTCTGGCTTTATAATTAG-3′; PRIB4:(SEQ ID NO: 5) 5′-GGGGGGCTCGAGCAATTCAAAAGTCATTCAGACTC-3′; KC176:(SEQ ID NO: 6) 5′-GAGGTGGAGCCCGGGGTCTGCTCGGCCCCAGGC-3′; ET-TCRF1:(SEQ ID NO: 7) 5′-CCCACCGGTCAGTCAGTGACGCAGCCC-3′;  KC-170:(SEQ ID NO: 8) 5′-GTGGAGTTCGAAAAGGTGACTTACGTTTGTCTGCTCGGCCCCA G-3′;KC231: (SEQ ID NO: 9) 5′CGATAAGTGTACTTACGTTTTCATTATTCCATCGGCATGTACTCTTCTTCCTCTCG-3′; KC208: (SEQ ID NO: 10) 5′GTGGAGATCGATAAGTGTACTTACGTTTTCATTATCGCGATCCGGAGTTAACGTCTGCTCGGCCCCAG-3′; KC327B: (SEQ ID NO: 11)5′-TAGGTGTCCGGAGCACCTACTTCAAGTTCTAC-3′; KC328B: (SEQ ID NO: 12)5′-TAGGTGTCGCGAAGTTAGTGTTGAGATGATG-3′; AP2: (SEQ ID NO: 13)5′-ACTCACTATAGGGCTCGAGCGGC-3′; Cα HYB: (SEQ ID NO: 14) 5′GCTGTCCTGAGACCGAGGATCTTTTAACTG 3′; Cβ HYB: (SEQ ID NO: 15)5′-TTGTTTGTTTGCAATCTGTGCTTTTGATGG-3′.

The TCR gene was cloned from the T cell clone A2.1 264#5. We designatethe single-chain TCR derived from this T cell clone 264scTCR. Poly(A)⁺RNA was extracted from the cells using a MicroFast Track kit(Invitrogen, Carlsbad, Calif.), and double stranded cDNA was preparedand ligated to a double stranded adaptor oligonucleotide using theMarathon cDNA Amplification Kit (Clontech, Palo Alto, Calif.). Toidentify the Vα and Vβ segments, 5′-RACE PCR was performed using theA2.1 264#5 cDNA preparation and above-listed primers AP2 (specific forthe adaptor DNA) and Cα HYB (specific for the constant domain of the achain) or Cβ HYB (specific for the constant domain of the β chain). PCRfragments were cloned into the pCR2.1 vector using the TA cloning kit(Invitrogen), and the sequence was determined using M13 forward andreverse primers. The T cell receptor Vα chain was amplified usingprimers KC228 and KC229 to produce an SfiI/SpeI fragment, and the VβCβchain was amplified using primers PRIB4 and KC176 to generate anXhoI/XmaI fragment. The Cβ chain was truncated just before the cysteineresidue at amino acid 127 of the full length Cβ chain. The SfiI/SpeI Vαchain fragment was subcloned into SfiI/SpeI digested pKC60, an E. coliexpression vector that encodes an irrelevant TCR, replacing the originalTCR insert. The XhoI/XmaI VβCβ fragment was then ligated into anXhoI/XmaI digest of this vector yielding a vector encoding a solublethree domain 264scTCR. The three domain T cell receptor from thisconstruct was amplified using primers ET-TCRF1 and KC170 to generate anAgeI/ClaI DNA fragment, which was then used as a template for PCR withprimers KC231 and KC208 to produce an AgeI/HpaI fragment.

The human IL-2 coding sequence was cloned by RT-PCR from total RNAisolated from Jurkat cells using a Mini Total RNA Kit (Qiagen, ValenciaCalif.) and Qiashredder (Qiagen, Valencia Calif.). Reverse transcriptionwas carried out using primer KC328B, and PCR was carried out usingprimers KC327B and KC328B to produce a BspEI/NruI human IL-2 fragment.The BspEI/NruI IL-2 fragment was cloned into BspEI/NruI digestedp149B1SP, a cloning vector encoding an irrelevant TCR/antibody fusionprotein, replacing the antibody portion of the fusion protein. The IL-2modified vector was digested with AgeI and HpaI and the AgeI/HpaI264scTCR fragment described above was ligated into it. Finally, anAgeI/ClaI 264scTCR/IL-2 fusion protein fragment was cloned intoAgeI/BstBI digested pSUN27, a scTCR/mouse kappa fusion vector, replacingthe irrelevant TCR originally cloned in the vector, yielding the264scTCR/IL-2 fusion protein expression vector, pSUN38. The264scTCR/kappa fusion used as a negative control for some of the flowcytometry analyses was generated by cloning an AgeI/BstBI 264scTCRfragment into AgeI/BstBI digested pSUN27, replacing the original TCR.

For production of fusion protein in mammalian cells, CHO.K1 cells wereelectroporated using a Bio-Rad Gene Pulser, followed by limitingdilution cloning and selection in medium containing 1 mg/mL G418.

4. Protein Purification

264scTCR/IL-2 was purified from cell culture supernatant fluid byimmunoaffinity chromatography using the monoclonal anti-murine TCRantibody H57-597, which recognizes an epitope in the constant region ofthe TCR β chain, coupled to a Sepharose 4B column (Amersham Pharmacia,Piscataway, N.J.). The purified sample was then concentrated andbuffer-exchanged into PBS using an Ultrafree-15 centrifugal filter witha 30 kDa molecular weight cutoff membrane (Millipore, Bedford, Mass.).The TCR fusion protein samples were stored at 2-8° C. (short term) or at−80° C. (long term) for biochemical and functional analysis. SDS-PAGEwas performed under either reducing or non-reducing conditions using4-12% Nu-PAGE polyacrylamide gels (Novex, San Diego, Calif.) and theNovex EX-Cell II system. SDS-PAGE gels were stained with Coomassie blue.

5. ELISA

All ELISAs were performed using Maxisorb 96 well plates (Nunc,Rochester, N.Y.) coated with 100-200 ng/well anti-human IL-2 mAb oranti-murine TCR Vβ3 mAb. Fusion protein was detected with biotinylatedanti-murine TCR H57 mAb, anti-murine TCR Vβ3 mAb, or anti-IL-2polyclonal Ab followed by streptavidin-HRP (Kirkegaard and PerryLaboratories, Gaithersburg, Md.), TMB substrate, and 0.18 M H₂SO₄ toquench the reaction (BioFX, Owings Mills, Md.). Absorbance was measuredat 450 nm using a 96 well plate reader (Bio-Tek Instruments, Inc.,Winooski, Vt.).

6. Cell Staining with TCR Fusion Proteins

T2 cells pulsed with either p53 (aa 149-157) or p53 (aa 264-272) peptidewere incubated with 0.5 μg of 264scTCR/IL-2 fusion protein in 1% FBS inPBS for 30 minutes at room temperature. The cells were then incubatedwith 0.5 μg anti-IL-2 Ab or 0.5 μg biotinylated anti-TCR H57-597 mAb for30 minutes at room temperature followed by 1 μg anti-murine kappa-PE or5 ng streptavidin-PE, respectively (both from Becton Dickenson, FranklinLakes, N.J.). Samples were washed with 1% FBS in PBS before FACScananalysis (Becton Dickenson, Franklin Lakes, N.J.). To determine if bothp53 peptides bound to HLA-A2 similarly, peptide loaded cells werestained with BB7.2 for 30 minutes at room temperature followed by FITClabeled goat anti-mouse IgG and analyzed on a FACScan instrument.

CTLL-2 cells were incubated with 0.5 μg of fusion protein for 30 minutesat room temperature. To detect the bound fusion protein, 0.5 μgbiotinylated anti-TCR V133 mAb was added and incubated for 30 minutes atroom temperature followed by incubation with 5 ng streptavidin-PE, orthe protein was detected using 0.5 μg PE-labeled HLA-A2.1 p53 (aa264-272) tetramer for 30 minutes. Conjugated HLA-A2 tetramers loadedwith p53 peptides were produced as described previously [1]. Sampleswere washed with 1% FBS in PBS before FACScan analysis. For IL-2receptor blocking experiments, CTLL-2 cells were incubated with α-humanCD25 blocking antibody or isotype control antibody for 30 minutes beforeincubation with 264scTCR/IL-2 or 264scTCR/kappa fusion protein. Forstaining of BALB/c mouse splenocytes, staining was carried out asdescribed for the CTLL-2 cells using HLA-A2.1 p53 (aa 264-272) tetramersto detect bound fusion protein.

A375 cells were harvested with enzyme-free cell dissociation buffer(Sigma, St. Louis, Mo.). Samples of 5×10⁵ cells were washed with 1% FBSin PBS and incubated with no fusion protein, 5 μg 3C8 (an irrelevantTCR/IL-2 fusion protein), or 5 μg 264scTCR/IL-2 for 30 minutes at roomtemperature followed by incubation with 1 μg biotinylated H57-597 mAb.Cells were then incubated with PE-labeled streptavidin for 15 minutes atroom temperature, washed, and analyzed by FACScan.

7. Cell Conjugation

T2 cells pulsed with either p53 (aa 264-272) peptide or p53 (aa 149-157)peptide were labeled with 7.88 ng/ml dihydroethidium (HE) (MolecularProbes, Inc., Eugene, Oreg.), and CTLL-2 cells were labeled with 50ng/ml calcein AM (Molecular Probes, Inc., Eugene Oreg.). After washing,the two populations of labeled cells were mixed together at a 1:1 ratioin the presence or absence of 2 μg 264scTCR/IL-2 fusion protein for 20minutes at room temperature. Cells were then analyzed by FACScan.

8. Bioassay

CTLL-2 cells were seeded at 4×10³ cells/well in 100 μl culture mediumcontaining various concentrations of either recombinant IL-2 or264scTCR/IL-2 and incubated for 21 hours at 37° C. and 5% CO₂. As acontrol for specificity CTLL-2 cells were incubated with 264scTCR/IL-2in the presence or absence of 5 or 50 μg anti-human CD25 blockingantibody or isotype control antibody and incubated for 21 hours at 37°C. and 5% CO₂. Cell proliferation reagent WST-1 (Roche Inc.,Indianapolis, Ind.) was added at 20 μl/well and incubated for 4 hours at37° C. and 5% CO₂. Absorbance was read at 450 nm on a 96-well platereader.

9. Pharmacokinetics in Mice

For all experiments involving animals, principles of laboratory animalcare (NIH publication No. 85-23, revised 1985) were followed, as well asspecific national laws where applicable. Female BALB/c mice wereinjected intravenously via the lateral tail vein with 32 264scTCR/IL-2fusion protein diluted with PBS to a total volume of 100 Serum wascollected from one group of mice not injected with 264scTCR/IL-2 toestablish background levels. Serum was collected by tail bleed from theinjected groups at 15 and 30 minutes, 1, 2, 4, 8, and 24 hours. Bloodsamples were centrifuged at 14,000×g at 4° C. for 10 minutes, and serumwas collected and stored at −80° C. until use. 264scTCR/IL-2concentrations were determined by ELISA using anti-TCR V133 or anti-IL-2monoclonal antibodies for capture and either biotinylated anti-TCR H57monoclonal or anti-IL-2 polyclonal antibodies followed by streptavidinHRP for detection.

10. In Vivo Studies

Female athymic nude mice (nu/nu) were injected with 5.0×10⁵ A375-C15Ncells via the lateral tail vein. Animals were injected with varyingdoses of either 264scTCR/IL-2 (32, 10, 3, 1, or 0.1 μg in 100 μl totalvolume) or recombinant human IL-2 (8, 2.5, 0.75, 0.25, or 0.025 μg in100 μl total volume) days 1, 2, 3, 4, 7, 10, 14, 17, 21, 28, and 35post-tumor cell injection. Forty-two days after tumor cell injection,all animals were humanely sacrificed, the lungs were removed and fixedin Bouin's solution, and surface pulmonary tumor nodules were counted.Tumor nodules on each lung were counted by two observers and the averagecounts were recorded.

11. TCR Constructs and Fusion Proteins Comprising IgG and Bir a TagSequence

The TCR gene was cloned from the T cell clone A2.1 264#5 as described.The single-chain TCR derived from this T cell clone was designated as264scTCR. The three domain single chain 264scTCR was amplified using a264scTCR/IL-2 fusion protein construct as a template. To generate the264scTCR/IgG1 expression construct, the single chain TCR fragment wasligated into an antibody heavy chain expression vector, replacing theantibody variable region and yielding a single chain TCR fused to ahuman IgG1 heavy chain region. To generate the 264scTCR/trunIgG1, theTCR fragment was ligated into an expression vector containing the IgG1heavy domain that was truncated prior to the hinge region that allowsdisulfide bonding.

To generate the 264scTCR/BirA expression construct, the single chain TCRfragment was ligated into an expression vector containing the BirA tagsequence (Beckett, D. et al. Protein Sci. 1999 April; 8(4):921-9), suchthat the tag sequence was expressed in frame at the C-terminus of the264scTCR molecule.

The Cytomegalovirus single-chain TCR (CMVscTCR) was cloned from CTLsstimulated with HLA-A2 restricted CMV-pp65 peptides. The IgG1 fragmentwas amplified from 264scTCR/IgG1 DNA to create the CMVscTCR/IgG1construct.

For production of the fusion proteins in mammalian cells, CHO.K1 cellswere electroporated using a Bio-Rad Gene Pulser, followed by limitingdilution cloning and selection in medium containing 1 mg/ml G418.

Protein purification was carried out as follows. 264scTCR/IgG1,264scTCR/BirA and 264scTCR/trunIgG1 were purified from cell culturesupernatant fluid by immunoaffinity chromatography using the H57-597monoclonal antibody coupled to a Sepharose 4B column (AmershamPharmacia, Piscataway, N.J.). CMVscTCR/IgG1 was purified from cellculture supernatant fluid by immunoaffinity chromatography using the BF1monoclonal antibody coupled to a Sepharose 4B column (AmershamPharmacia, Piscataway, N.J.). 264scTCR/BirA was biotinylated withbiotin-protein ligase (Avidity) under conditions recommended by themanufacturer.

12. Detection of Cell Staining by 264scTCR Reagents by Flow Cytometry

The ability of 264scTCR reagents to stain fixed and unfixed cells wascharacterized in several studies. Cell staining strategies included useof 264scTCR fusions carrying various detectable domains, and detectingthe cellular interaction of these fusions with various fluorescentlylabeled probes. Several controls were used to assess specific staining.Controls included staining cells that lacked the p53(aa264-273) antigenwith the 264scTCR reagents, staining p53-positive cells with theCMVscTCR reagents, staining p53-positive cells with secondary stainingreagents alone, and staining p53-positive cells with the 264scTCRreagents with and without competitive blocking reagents such as solubleHLA-A2/p53 multimers.

Monomeric or multimeric forms of the 264scTCR were tested for theirability to specifically stain cells. T2 cells were loaded with p53(aa264-273) or p53 (aa149-157) at 100 μg/ml for 2.5 hour at 37° C. Aftera wash step to remove excess peptide, the cells were incubated with264scTCR/IL-2, 264scTCR/IgG1, 264scTCR/trIgG1 or 264scTCR/BirA (withoutbiotinylation) at 125 pM for 30-45 minutes. SDS-PAGE analysis of reducedand non-reduced samples indicated that the 264scTCR/trunIgG1 and264scTCR/BirA proteins are monomeric and the 264scTCR/IgG1 protein is adimer. After another wash step, cells were incubated for 30 minutes with2.5 μg PE-conjugated H57 mAb (H57-PE). The cells were washed andanalyzed on a FACScan flow cytometry instrument (BD Sciences, San Jose,Calif.) using CellQuest software (BD Biosciences, San Jose, Calif.).Unstained and H57-PE stained T2 cells were also analyzed to establishbackground staining.

The following documents are referred to (by a number as shown below)throughout the present disclosure. Each document is incorporated byreference.

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The disclosures of all references mentioned herein are incorporatedherein by reference. The invention has been described with reference topreferred embodiments thereof. However, it will be appreciated thatthose skilled in the art, upon consideration of this disclosure, maymake modifications and improvements within the spirit and scope of theinvention.

What is claimed is:
 1. A method for detecting cells or tissue comprisinga peptide antigen presented on the cells or tissue in the context of anMHC complex, the method comprising: a) contacting the cells or tissuewith at least one soluble TCR molecule or functional fragment thereofunder conditions that form a specific binding complex between thepresented peptide antigen and the soluble TCR or fragment, b) washingthe cells or tissue under conditions appropriate to remove any solubleTCR molecule or fragment not bound to the presented peptide antigen; andc) detecting the specific binding complex as being indicative of cellsor tissue comprising the presented peptide antigen.
 2. The method ofclaim 1, wherein the cells or tissue are further contacted with at leastone blocking agent.
 3. The method of claim 2, wherein the method furthercomprises contacting the cells or tissue with the blocking agent beforestep a) to reduce non-specific binding between the soluble TCR orfragment and the cells.
 4. The method of claim 2, wherein the blockingagent is a peroxide, serum protein, antibody, or fragment thereof. 5.The method of claim 1, wherein the method further comprises contactingthe complex with a competing MHC molecule or fragment thereof underconditions that compete with and specifically bind the soluble TCR orfragment bound to the complex.
 6. The method of claim 5, whereinessentially all of the soluble TCR or fragment is bound to the competingMHC molecule or fragment thereof to form a competition complex.
 7. Themethod of claim 6, wherein the method further comprises detecting thecompetition complex and determining the binding specificity of the MHCmolecule or the soluble TCR.
 8. The method of claim 5, wherein the MHCmolecule or fragment is single-chain.
 9. The method of claim 5, whereinthe MHC molecule or fragment is loaded with peptide antigen.
 10. Themethod of claim 1, wherein the method further comprises contacting thecells or tissue with at least one denaturing agent.
 11. The method ofclaim 10, wherein the method further comprises contacting the cells ortissue with the denaturing agent before step a) and denaturing (fixing)the cells or tissue.
 12. The method of claim 1, wherein the methodfurther comprises placing a plurality of cells or tissue in an array.13. The method of claim 12, wherein the method is performed in eachelement of the array comprising cells or tissue.
 14. The method of claim12, wherein the method is performed substantially simultaneously in eachelement of the array.
 15. The method of claim 12, wherein step c)further comprises scanning the array and generating image signalsindicative of presence of the specific binding complex.
 16. The methodof claim 15, wherein step c) further comprises outputting the signals inreal-time to a user and optionally indexing stored images of the imagesignal.
 17. The method of claim 1, wherein the amount of any peptideantigen present on the cells is less than about 100,000 copies.
 18. Themethod of claim 17, wherein the amount of any peptide antigen present onthe cells is less than about 400 copies.
 19. The method of claim 1,wherein the peptide antigen is a tumor-associated peptide antigen. 20.The method of claim 1, wherein the cells or tissue are suspended. 21.The method of claim 1, wherein the soluble TCR molecule or fragmentcomprises at least one single-chain TCR.
 22. The method of claim 1,wherein the soluble TCR molecule or fragment is detectably-labeled. 23.The method of claim 22, wherein the detectable label is biotin,streptavidin, an enzyme or catalytically active fragment thereof, aradionuclide, a nanoparticle, a paramagnetic metal ion, or afluorescent, phosphorescent, or chemiluminescent molecule.
 24. Themethod of claim 21, wherein the single-chain TCR or fragment furthercomprises at least one covalently bound cytokine.
 25. The method ofclaim 24, wherein the soluble TCR molecule or fragment is a single-chainTCR comprising a covalently bound cytokine or fragment thereof.
 26. Themethod of claim 25, wherein the single-chain TCR or fragment comprisessequence encoding interleukin-2 (IL-2).
 27. The method of claim 21,wherein the single-chain TCR or fragment further comprises at least onecovalently bound immunoglobin domain.
 28. The method of claim 27,wherein the soluble TCR molecule or fragment is a single-chain TCRcomprising a covalently bound immunoglobin domain or fragment thereof.29. The method of claim 28, wherein the single-chain TCR or fragmentcomprises sequence encoding an IgG1 domain or fragment thereof.
 30. Themethod of claim 1, wherein the MHC complex is HLA-A2 restricted.
 31. Themethod of claim 1, wherein the method further comprises performing acontrol to detect any binding between the soluble TCR or fragment tocells that do not comprise the peptide antigen.
 32. The method of claim1, wherein the presented peptide antigen is p53 (aa 149-157) or p53 (aa264-272).
 33. The method of claim 1, wherein the method is performed invivo, ex vivo, or in vitro.
 34. The method of claim 1, wherein the cellor tissue is a tumor cell or tissue.
 35. A method for detecting a tumorcell or tissue in a subject, wherein the cell or tissue comprises atumor-associated peptide antigen presented on the cells or tissue in thecontext of an MHC complex, the method comprising: a) administering tothe subject a soluble TCR molecule or functional fragment thereof underconditions that form a specific binding complex between the presentedpeptide antigen and the soluble TCR molecule or fragment; and b)detecting the specific binding complex as being indicative of a tumorcell or tissue comprising the presented tumor-associated peptideantigen.
 36. The method of claim 35, wherein the soluble TCR molecule orfragment comprises at least one single-chain TCR.
 37. The method ofclaim 35, wherein the soluble TCR molecule or fragment isdetectably-labeled.
 38. The method of claim 37, wherein the detectablelabel is biotin, streptavidin, an enzyme or catalytically activefragment thereof, a radionuclide, a nanoparticle, a paramagnetic metalion, or a fluorescent, phosphorescent, or chemiluminescent molecule.